Compositions and methods for removing detergent from aqueous solutions
By adding a second surfactant or detergent to the aqueous solution to form a mixed micelle, and using a size-based separation method, the problem of difficulty in removing detergent and surfactant in the prior art is solved, and the function of retaining protein while removing is achieved.
Patent Information
- Application Number
- CN202510210892.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2019-03-14
- Filing Date
- 2020-03-13
- Publication Date
- 2025-05-30
AI Technical Summary
The prior art is difficult to effectively remove detergents and surfactants from aqueous solutions, especially in pharmaceutical preparations, which can interfere with the analytical method and are difficult to remove completely.
The concentration of the second surfactant or detergent is directly added to the solution to reach or exceed the critical micelle concentration, forming a mixed micelle including two surfactant/detergent and removing proteins using size-based separation methods such as ultrafiltration and gel filtration to remove the stain remover and surfactant.
Effective removal of detergent and surfactant from aqueous solution while retaining functional proteins and peptides, reducing interference to subsequent analytical methods.
Smart Images
Figure CN120058833A_ABST
Abstract
Description
[0001] This invention is a divisional application of the application with the filing date of March 13, 2020, application number 202080033968.8, and invention title "Compositions and Methods for Removing Detergents from Aqueous Solutions".
[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 818,554, filed on March 14, 2019. All of these and all other cited external materials are hereby incorporated by reference in their entirety. If the definition or usage of a term in the incorporated reference is inconsistent with or contrary to the definition of the term provided herein, the definition of the term provided herein shall control. TECHNICAL FIELD
[0003] The field of the present invention is the removal of detergents or surfactants, particularly from pharmaceutical formulations. BACKGROUND ART
[0004] The following description includes information that may be helpful in understanding the present invention. This is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, nor is it an admission that any specifically or implicitly cited publication is prior art.
[0005] Excipients are often added to small molecule (e.g., <500 D) and / or protein solutions (such as pharmaceutical formulations) to reduce aggregation, prevent non-specific binding to container surfaces, and otherwise improve stability. Traditional excipients include proteins (such as human or bovine serum albumin, ovalbumin, immunoglobulins, etc.), sugars and polysaccharides, and soluble polymers (such as polyvinylpyrrolidone). To provide non-immunogenic formulations, nonionic and zwitterionic detergents and surfactants are increasingly being used for this purpose. However, many detergents and surfactants interfere with analytical methods (such as immunoassays, peptide-based assays, cell-based assays, mass spectrometry, etc.) used for the characterization and / or quality control of such solutions.
[0006] Unfortunately, once detergents and surfactants are introduced, they are difficult to remove from solution. Ion exchange can be used to remove charged (e.g., cationic or anionic) detergents and surfactants, but many commonly used surfactants do not carry a formal charge. Hydrophobic media can bind substantial amounts of detergents and / or surfactants; however, such hydrophobic media can also bind substantial amounts of the target protein (which may be present at low concentration). Dialysis is used to remove detergents or surfactants, but with only partial success because the self-association of the detergent or surfactant into micelles reduces the amount of "solubilized" surfactant or detergent that can be removed by dialysis. Thus, the rate of removal of detergents and surfactants by concentration gradient-driven dialysis is very slow. The formation of micelles also interferes with the removal of surfactants and detergents by more active size-based separation methods such as gel filtration and ultrafiltration because their size causes them to be retained by ultrafiltration membranes and excluded from the internal volume of gel filtration media.
[0007] Commercial products are available for removing surfactants and detergents from aqueous solutions. For example, the HiPPR resin from ThermoFisher claims to remove 95% of surfactants or detergents from low-concentration protein / peptide solutions while maintaining the protein / peptide content. G Biosciences offers a detergent resin that is described as having a high affinity for most surfactants and a low affinity for most proteins and peptides. Calbiochem offers a hydrophobic CALBIOSORB TM resin for batch removal of surfactants and recommends using this resin in dialysis buffers to avoid non-specific binding of the relevant proteins. However, it is not clear whether these products can effectively remove polysorbate surfactants, which are increasingly used as excipients, or whether all proteins / peptides remain in solution after treatment.
[0008] In some applications, peptide solutions are extracted using an organic solvent such as ethyl acetate. In such extractions, relatively hydrophobic detergents transfer to the relatively immiscible organic layer of the extraction mixture. However, because of the potential for irreversible denaturation, such methods typically involve subsequent characterization of the physical properties of the protein (e.g., by mass spectrometry), which may not provide information about activity.
[0009] Accordingly, there remains a need for a simple and convenient method for removing detergents and / or surfactants from aqueous solutions while retaining functional proteins and / or peptides in solution. SUMMARY OF THE INVENTION
[0010] The compositions and methods of the present inventive concept provide a method for removing a surfactant or a detergent (e.g., a galactoside detergent such as octyl-β-galactoside; a glucoside detergent such as N-octanoyl-N-methylglucamine (MEGA 8), N-nonanoyl-N-methylglucamine (MEGA 9), and / or N-decanoyl-N-methylglucamine (MEGA10); a cholamide detergent such as 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate inner salt (CHAPS), 3-[3-(cholamidopropyl)dimethylamino]-2-hydroxy-1-propanesulfonate (CHAPSO), and / or (3a,5b,7a,12a)-N,N-bis[3-(D-gluconamido)propyl]-3,7,12-trihydroxycholestane-24-amine (BIGCHAPS); and / or a sulfobetaine detergent such as sulfobetaine 3-10) from a protein-containing solution by directly adding a second surfactant or a detergent to the solution, such that the added concentration of the second surfactant or detergent is at least equal to the critical micelle concentration of the second surfactant or detergent, allowing the formation of mixed micelles comprising two surfactants / detergents, and separating the protein from the resulting mixed micelles using a size-based separation method (such as ultrafiltration and / or gel filtration). Some embodiments include the step of blocking the ultrafiltration membrane used in the ultrafiltration separation before or at the time of separating the protein from the mixed micelles. Some embodiments include the step of blocking the gel filtration medium used in the gel filtration separation before or at the time of separating the protein from the mixed micelles. Some embodiments of the present inventive concept include the step of collecting an analytical fraction containing the protein from the size-based separation method, and the analytical fraction can be analyzed using a cell-based assay. The protein can be botulinum neurotoxin (e.g., Botulinum neurotoxin type A or BoNT / A). BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 : Figure 1 Schematically depicts an exemplary process for removing a detergent from a protein-containing solution.
[0012] Figure 2 : Figure 2 Shows CMC 535 TM Typical results characterizing the detergent content in aqueous solutions treated with different first and second detergents.
[0013] Figures 3A to 3C : Figure 3A Shows typical results of removing polysorbate 20 from a solution by diafiltration in the presence of different second detergents. Figure 3BIllustrates typical results of removing polysorbate 80 from a solution by diafiltration in the presence of different second detergents. Figure 3C Illustrates typical results of removing Triton X-100 from a solution by diafiltration in the presence of different second detergents.
[0014] Figures 4A to 4C : Figure 4A Illustrates typical results of botulinum neurotoxin type A (BoNT / A) obtained from a solution by the method of the present inventive concept. The upper small figure illustrates BoNT / A recovered from a BoNT / A sample containing polysorbate 20. The lower small figure illustrates data normalized relative to a control sample containing BoNT / A and no first detergent (polysorbate 20). Figure 4B Illustrates typical results of botulinum neurotoxin type A (BoNT / A) obtained from a solution by the method of the present inventive concept. The upper small figure illustrates BoNT / A recovered from a BoNT / A sample containing polysorbate 80. The lower small figure illustrates data normalized relative to a control sample containing BoNT / A and no first detergent (polysorbate 80). Figure 4C Illustrates typical results of botulinum neurotoxin type A (BoNT / A) obtained from a solution by the method of the present inventive concept. The upper small figure illustrates BoNT / A recovered from a BoNT / A sample containing Triton X-100. The lower small figure illustrates data normalized relative to a control sample containing BoNT / A and no first detergent (Triton X-100).
[0015] Figure 5A and Figure 5B : Figure 5A Illustrates with CMC 535 TM Typical results of a study on the effect of additional BigCHAP washings on removing polysorbate 80 as determined by the method. Figure 5B Illustrates with CMC 535 TM Typical results of a study on the effect of additional buffer washings on removing polysorbate 80 as determined by the method.
[0016] Figure 6A and Figure 6B : Figure 6A Illustrates typical results of a study on recovering BoNT / A from a sample containing polysorbate 80, which is subsequently extracted with mixed micelles containing BigCHAP and removed by repeated washing with a buffer containing BigCHAP. The upper small figure illustrates the results relative to BoNT / A added to the sample. The lower small figure illustrates the results of the recovery rate relative to a BoNT / A sample without polysorbate 80. Figure 6BShows typical results of a study on the recovery of BoNT / A from samples containing polysorbate 80, which was subsequently extracted with micelles mixed with BigCHAP and removed by repeated washing with a buffer without a second detergent. The small graph on the left shows the results relative to the BoNT / A added to the sample. The small graph on the right shows the results of the recovery rate of the BoNT / A sample without polysorbate 80.
[0017] Figures 7A to 7C : Figure 7A Shows typical results of the extraction of polysorbate 20 from BoNT / A samples with different concentrations of octyl-β-galactoside (OBG), BigCHAP, or CHAPS, as characterized by the CMC 535 TM method. Figure 7B Shows typical results of the extraction of polysorbate 80 from BoNT / A samples with different concentrations of octyl-β-galactoside (OBG), BigCHAP, or CHAPS, as described by the CMC 535 TM method. Figure 7C Shows typical results of the extraction of Triton X-100 from BoNT / A samples with different concentrations of octyl-β-galactoside (OBG), BigCHAP, or CHAPS, as characterized by the CMC 535 TM method.
[0018] Figures 8A to 8C : Figure 8A Shows typical results of a study on the recovery of BoNT / A from samples containing polysorbate 20 after mixed micelle extraction with different concentrations of octyl-β-galactoside (OBG), BigCHAP, or CHAPS, as characterized by using Biosentel BoTestA / E reagent. The upper small graph shows the percentage of the BoNT / A recovery rate relative to the added amount. The lower small graph shows the BoNT / A recovery rate, which is the percentage of the recovery rate of the control BoNT / A sample without polysorbate 20. Figure 8B Shows typical results of a study on the recovery of BoNT / A from samples containing polysorbate 80 after mixed micelle extraction with different concentrations of octyl-β-galactoside (OBG), BigCHAP, or CHAPS, as characterized by using BioSentinel BoTestA / E reagent. The upper small graph shows the percentage of the BoNT / A recovery rate relative to the added amount. The lower small graph shows the BoNT / A recovery rate, which is the percentage of the recovery rate of the control BoNT / A sample without polysorbate 80. Figure 8CShows typical results of a study on the recovery of BoNT / A from samples containing Triton X-100 after mixed micelle extraction with different concentrations of octyl-β-D-galactopyranoside (OBG), BigCHAP, or CHAPS, as characterized by BoTestA / E TM The upper small graph shows the percentage of BoNT / A recovery relative to the added amount. The lower small graph shows the BoNT / A recovery, which is the percentage of the recovery of a control BoNT / A sample without Triton X-100.
[0019] Figures 9A to 9D : Figure 9A Shows typical results obtained from a cell-based BoNT / A assay using BoNT / A prepared in a surfactant-free medium or a medium containing the polysorbate 80 excipient. Figure 9B Shows Figure 9A Typical results of a similar study in which the BoNT / A sample was dialyzed before the assay. Figure 9C Shows typical results of a study on the removal of the polysorbate 80 excipient from a sample containing BoNT / A using a second surfactant, specifically the effect of the polysorbate 80 excipient and residual surfactant in the sample extracted with the second surfactant on the cell morphology in the Bioscontinel BoTestA / E TM assay. Figure 9D Shows typical results of the EC50 determination of BoNT / A in the Biosontinel BoTestA / E TM assay using BoNT / A prepared in a surfactant-containing medium and BoNT / A prepared in a medium containing the polysorbate 80 excipient and subjected to mixed micelle extraction.
[0020] Figure 10 : Figure 10 Shows typical results of a study on the retention of mixed micelles of polysorbate 80 and BigCHAP by Amicon regenerated cellulose ultrafiltration membranes with different molecular weight cutoffs (MWCs).
[0021] Figure 11 : Figure 11 Shows a comparison of the retention rates of mixed micelles of polysorbate 80 and BigCHAP by PES (Pierce, Sartoris) ultrafiltration membranes with different MWCs.
[0022] Figure 12 : Figure 12Shows typical results of the recovery study of BoNT / A after removing mixed micelles of polysorbate 80 with regenerated cellulose (Amicon) and PES (Pierce, Sartoris) ultrafiltration membranes with different molecular weight cut-offs.
[0023] Figure 13 : Figure 13 Shows typical results of the study of the effects of pre-blocking with human serum albumin (HSA) and combined pre-blocking and sample spiking on the recovery of the first detergent and BoNT / A after separation of mixed micelles.
[0024] Figure 14 : Figure 14 Shows Biosentel in vivo BoCell TM Typical results of the study of the effects of pre-blocking with human serum albumin (HSA) and combined pre-blocking and sample spiking on cells in the determination. Detailed Description
[0025] The subject matter of the present invention provides compositions and methods wherein a first detergent or surfactant (e.g., a detergent or surfactant added as an excipient to a protein or peptide solution) is at least partially removed by directly adding a second detergent or surfactant at a concentration exceeding the critical micelle concentration (CMC) of the second detergent or surfactant. The second detergent or surfactant can be selected to form micelles having a low molecular weight (e.g., less than about 500 kD, 200 kD, 150 kD or 100 kD) and / or a small hydrodynamic radius (e.g., less than the hydrodynamic radius of the target protein). In a preferred embodiment, the CMC of the second detergent of the surfactant is from about 3.5 mM to about 40 mM, and / or forms micelles having an average molecular weight in the range of about 8 kD to about 40 kD. Typical first detergents / surfactants include but are not limited to polysorbate 20, polysorbate 80 and Triton X-100. Suitable second detergents or surfactants can be ionic, non-ionic or zwitterionic. Typical second detergents / surfactants include but are not limited to galactoside detergents (e.g., octyl-β-galactoside), glucamine detergents (e.g., MEGA 8, MEGA 9, MEGA 10), cholamide detergents (e.g., CHAPS, CHAPSO, BIGCHAPS) and sulfobetaine detergents (e.g., sulfobetaine 3-10).
[0026] In the present application, the term "about" defines a range of 20% of the nominal value.
[0027] A second detergent or surfactant can be selected such that it does not interfere or provides an acceptable level of interference (e.g., a level of interference that does not interfere with accurate characterization) with subsequent assays of the protein or peptide (e.g., immunoassays and / or cell-based assays). When the second detergent or surfactant is added directly to a solution containing the first detergent or surfactant, the molecules of the first detergent or surfactant are incorporated into the micelles of the second detergent or surfactant to form mixed micelles containing two surfactant / detergent species. Surprisingly, the inventors have found that the second detergent / surfactant species and its concentration can be selected to form relatively small mixed micelles. Specifically, the second detergent or surfactant can be selected such that when added directly to a solution containing the first detergent or surfactant, it results in the formation of mixed micelles having an effective hydrodynamic radius smaller than the protein or peptide to be analyzed (e.g., botulinum neurotoxin) and present in the initial solution. It should be understood that the surfactants and / or detergents used can be present as excipients to reduce aggregation of protein therapeutics in many pharmaceutical formulations.
[0028] The resulting small mixed micelles can be effectively separated and / or separated from the target protein or analyte by size-based separation methods (e.g., ultrafiltration, gel filtration, etc.), thereby removing at least a portion of the first and second detergents from the protein-containing solution. It should be understood that in some embodiments, e.g., those incorporating ultrafiltration, the target protein or peptide can be concentrated with reference to the concentration in the original sample volume during the detergent / surfactant removal process. In a preferred embodiment of the inventive concept, sufficient first detergent / surfactant and second detergent / surfactant are removed to reduce or eliminate interference with downstream analytical methods for characterizing protein quality and / or activity. The resulting high molecular weight fraction of the protein or peptide (e.g., the retentate or flow-through fraction) can then be characterized by methods that might be interfered with by the first and / or second surfactant or detergent.
[0029] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. The members of each group can be mentioned and claimed individually or in combination with other members of the group or other elements found herein. For convenience and / or patentability reasons, one or more members of a group can be included in or deleted from a group. When any such inclusion or deletion occurs, the specification is deemed to include the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.
[0030] In some embodiments, the mixed micelles of the inventive concept can pass through a membrane that retains botulinum neurotoxin (e.g., an ultrafiltration or dialysis membrane), removing the first surfactant from the botulinum neurotoxin solution while retaining the active protein. Similarly, the mixed micelles of the inventive concept can enter the internal volume of a suitably selected size exclusion chromatography medium, where the target protein or peptide in solution is excluded and elutes in the excluded (i.e., flow-through) volume and / or early fractions during size exclusion column chromatography.
[0031] In some embodiments, for example when the concentration of the protein or peptide to be characterized is low, the surface of the membrane or gel filtration medium can be blocked before and / or during the separation step. For example, such surfaces can be blocked by adding one or more exogenous proteins that do not interfere with downstream characterization methods (e.g., human serum albumin, bovine serum albumin, avian serum albumin, acetylated albumin, ovalbumin, mammalian immunoglobulins, avian immunoglobulins, mammalian or teleost gelatin, casein, etc.). In some embodiments, such surfaces can be blocked by adding long-chain polymers (e.g., polyvinylpyrrolidone or polyethylene glycol). This blocking can reduce or eliminate losses due to denaturation of the analyte on the separation surface and / or losses due to non-specific binding to the separation surface. The blocking protein and / or polymer can be applied at a concentration in the range of 0.1% to 10% (w / v), and can be applied before, during, or both before and during the separation step. In some embodiments, the blocking protein and / or polymer can be applied to the surface of the size separation membrane or medium before use, blocking the surface for a given time, and removing excess material by rinsing or washing before applying the sample containing the analyte.
[0032] Figure 1 An example of the method of the inventive concept is provided in the schematic illustration, which depicts the separation of mixed micelles from a solution containing botulinum neurotoxin (BoNT) using an ultrafiltration membrane spin column. As shown, a second detergent is added directly to the solution containing BoNT (which also contains the first detergent), thereby forming mixed micelles with a reduced hydrodynamic radius relative to micelles formed solely by the first detergent. Centrifugation in a spin column equipped with an ultrafiltration membrane of an appropriate molecular weight cut-off causes the mixed micelles to pass through the membrane and retain the BoNT. The retained BoNT-containing solution can be easily recovered by inverting the sample-containing portion of the spin column and performing a brief centrifugation. It should be appreciated that recovering the BoNT in a smaller volume than the applied sample can result in a BoNT concentration that facilitates analysis (e.g., using a cell-based assay or immunoassay).
[0033] The inventors have found that the method of the present inventive concept can effectively remove a first surfactant that interferes with biochemical characterization methods and cell-based assay methods. The inventors believe that the method of the present inventive concept can be similarly applied to physical methods such as mass spectrometry and capillary electrophoresis.
[0034] In a typical surfactant / detergent removal protocol, an Amicon UFC centrifugal filter unit is pre-blocked with a blocking buffer containing 0.5% w / v human serum albumin (HSA), and the residual blocking buffer is removed by centrifugation. Then, 100 μL of a sample or drug (e.g., 100 μM botulinum neurotoxin type A (BoNT / A) in 50 mM HEPES, 140 mM NaCl, 0.5% HSA (human serum albumin), and 0.1% first detergent) is mixed with 400 μL of a buffer containing a second detergent, and the concentration of the second detergent after mixing is selected to match or exceed the critical micelle concentration (CMC) of the second detergent (e.g., about 1% w / v or higher). In some embodiments, HSA can be omitted as HSA has been found to reduce the recovery of some BoNTs (e.g., BoNT / A).
[0035] After a short incubation period (e.g., about 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 1 hour), the mixture is applied to the centrifugal filter unit and centrifuged. In some embodiments, the centrifugal filter unit can be washed by adding 500 μL of a buffer containing the second detergent and applying a centrifugal force. After washing the centrifugal filter unit with 500 μL of wash buffer, the centrifugal filter unit is inverted and centrifuged briefly to recover a volume of about 15 μL that represents the detergent-extracted sample. This volume can be diluted for analysis and / or split between different analytical methods.
[0036] As Figure 2 shown, the inventors have found that a commercial product for characterizing detergent concentration (CMC 535 TM ) does not react with many detergents that have been found to be useful as the second detergent. As shown, the CMC 535 TM assay produced dose / response curves for polysorbate 20 (P20), polysorbate 80 (P80), and Triton X-100 (X100), but did not show a significant response to many potential second detergents within the tested concentration range.
[0037] Typical results of removing the first detergent using the above protocol and characterization using CMC 535 TM are as Figures 3A to 3CAs shown, octyl-β-glucoside, BigCHAP, and CHAPS can effectively remove polysorbate detergents. The removal of Triton X-100 seems to be independent of the choice of the second detergent.
[0038] Samples containing BoNT / A in a buffer with a first surfactant were treated similarly and characterized for BoNT / A content using the BioSentinel BoTest TM activity assay. The BoTest TM activity assay utilizes a reporter peptide that contains an analogue of the BoNT / A substrate and bears a pair of FRET fluorophores separated by the substrate peptide. Proteolysis results in the separation of the FRET pair and a distinct change in fluorescence. By mixing 50 μL of the extraction sample with 100 μL of 0.25 μM BoTest TM A / E reporter peptide substrate and quantifying with a dose / response curve, an in vitro BoTest TM assay was performed. The results are as Figures 4A to 4C shown. It should be understood that in some cases, the first detergent can activate BoNT / A to some extent. Thus, control samples retaining the first detergent may show a falsely elevated BoNT / A activity, creating the impression of a low recovery after removal of the mixed micelles of the first detergent in the test samples. Similarly, if the control provided does not contain the first detergent, the recovery rate may be overestimated. However, the use of certain second detergents can significantly improve the recovery of BoNT / A. As shown, the choice of the second detergent may affect the recovery of BoNT / A. For example, the detergent SB-10 may be incompatible with the BoTest TM assay.
[0039] In some embodiments, an additional volume of buffer containing a second detergent (BigCHAP) can be added to a sample containing BoNT / a and a first detergent (polysorbate 80), followed by additional rounds of centrifugation. As Figure 5A shown, as in the CMC35 TM assay, this can effectively further remove the first detergent. Figure 5B The effect of a similar study with a buffer without a second surfactant is shown. As shown, additional washing with a buffer containing a second detergent can remove additional first detergent not removed in the first mixed micelle extraction. Using a buffer for additional washing does not produce this effect.
[0040] It should be understood that improving the extraction of the initial detergent does not necessarily indicate an improvement in the recovery performance of the functional analyte. The samples evaluated above in Figure 5A and 5B were also characterized using the BoTestTMA / E reagent as described above. The results are inFigure 6A and Figure 6B is shown in. As shown, the recovery of functional BoNT / A decreases with additional washes. This may be due to non-specific binding resulting in denaturation and / or loss of BoNT / A. Thus, the mixed micelle extraction protocol may be applicable for optimal recovery of functional analytes, which may or may not be related to the most effective protocol for removing the primary detergent. In some embodiments, additional washes after the initial mixed micelle extraction may be omitted.
[0041] Figures 7A to 7C shows the results of an optimized mixed micelle extraction protocol developed for BoNT / A samples containing the first detergents polysorbate 20, polysorbate 80, and Triton X-100 and using octyl-β-galactoside, BigCHAP, and CHAPS as the second detergents, as characterized by CMC 535 TM (i.e., detecting residual first detergent). The centrifugal adsorption columns were pre-blocked with human serum albumin before use; human serum albumin was also added to the BoNT / A preparation. Surprisingly, the inventors found that some of the first detergents (e.g., Triton X-100) seemed to be able to be removed from the sample by diafiltration without forming mixed micelles. As shown, as determined by CMC 535 TM there was no significant difference in the effectiveness of removing the first detergent with 1% (w / v) and 5% (w / v) of the second detergent.
[0042] Figures 8A to 8C shows the results of an optimized mixed micelle extraction protocol developed for BoNT / A samples containing the first detergents polysorbate 20, polysorbate 80, and Triton X-100 and using octyl-β-galactoside, BigCHAP, and CHAPS as the second detergents, as characterized by BoTestA / E assay. The centrifugal adsorption columns were pre-blocked with human serum albumin before use; human serum albumin was also added to the BoNT / A preparation. Surprisingly, the inventors found that some of the first detergents (e.g., Triton X-100) seemed to be able to be removed from the sample by diafiltration without forming mixed micelles. As shown, for most first / second detergent combinations, the recovery of functional BoNT / A increased with increasing concentration of the second detergent.
[0043] Although BoTest TMThe A / E reagent is a solvated synthetic peptide that was used as a biochemical assay in the above study to characterize the recovery of BoNT / A. However, in some applications, cell-based assays can be used to characterize samples containing an analyte in which a first detergent has been removed by mixed micelle extraction. Such cell-based assays have the advantage of providing an additional selective step in the form of selective uptake of the analyte. For example, botulinum neurotoxins (such as BoNT / A) are selectively taken up by cultured motor neurons and some neuron-derived cells. The first detergent used as an excipient can have an adverse effect on such cells.
[0044] Figure 9A An example of such interference is shown. Samples of BoNT / A holotoxin purified in surfactant-free medium and a commercial product of a similar concentration of BoNT / A in medium containing polysorbate 80 (P80) excipient were applied to BioSentinel BoCell TM A / E cell-based assay that utilizes genetically modified cells expressing a BoNT / A-sensitive reporter peptide containing a pair of FRET fluorophores. The results of the emission ratio of these fluorophores relative to the logarithm of the BoNT / A concentration are as Figure 9A shown. As shown, the cell-based assay is essentially non-responsive to BoNT / A samples containing surfactants. Figure 9B Results of a similar study are shown where the samples were dialyzed before application to the cells. As shown, dialysis had little improvement. Although an EC 50 can be obtained by dialyzing the sample, when applied to the same assay, the estimated value is approximately 10-fold higher than that of BoNT / A provided in various surfactant-free media.
[0045] Therefore, the utility of mixed micelle extraction in reducing the first detergent effect on cells in culture was characterized using the cells and medium used in the BoCell TM A / E assay, and BoNT / A samples in iBAM2 cell medium containing 0.1% polysorbate 80 surfactant excipient. Aliquots (100 μL) of this BoNT / A formulation were extracted with 400 μL of 2% MEGA8 or 1% of other second surfactants and the resulting mixture was concentrated using Amicon Ultra TM centrifugal filters. The resulting concentrate was washed with an additional 450 μL of the second surfactant solution by an additional round of centrifugation, and then with an additional 450 μL of iBAM2 medium. The resulting sample (about 15 μL) was then adjusted to a volume of 100 μL with iBAM2 medium and added to BoCell T A / E cells in the wells of a culture plate. The cells were incubated at 37 °C, 5% CO 2Characterization and imaging were performed after 24 hours and 48 hours. The results at 48 hours are as Figure 9C shown. In Figure 9C , cells that were visually similar to the control cells were represented by stars. As shown, the results for MEGA 10, BIGCHAPS, and CHAPS were similar to those of untreated control cells and cells treated only with BAM2 medium. The inventors believe that the maintenance of normal cell distribution and morphology indicates successful removal of the polysorbate 80 excipient and maintenance of cell health.
[0046] In media containing polysorbate 80, studies were conducted with samples containing different concentrations of BoNT / A, treated in the same manner with BIGCHAPS as the second surfactant, and applied to the BioSentinel BoCell TM BoNT A / E cell-based assay. The results were compared with those obtained with BoNT / A in surfactant-free media, as Figure 9D shown. As shown, after mixed micelle extraction, the surfactant-containing samples provided dose / response curves in the cell-based BoNT / A assay that were very similar to those obtained from surfactant-free samples. Table 1 provides a comparison of the EC50 values obtained.
[0047]
[0048] Table 1
[0049] Overall, the average EC 50 obtained from the surfactant-containing samples was 50.5% (CV 4.8%) of the average EC 50 obtained from the control samples and could be corrected with an adjustment factor (e.g., 0.5).
[0050] In the above studies, the molecular weight cut-off (MWC) of the ultrafiltration membrane used was 100 kDa. However, it should be understood that due to the shape of the protein molecules and the distribution of the membrane pore sizes, proteins with slightly higher molecular weights (e.g., 150 kDa BoNT / A) may pass through such membranes. Therefore, further studies were conducted to characterize the effective range of MWC for removing surfactants from the mixed micelle solution. The passage of polysorbate 80 (as the first detergent) in the presence or absence of BIGCHAPS (as the second detergent) was studied. The materials passing through the regenerated cellulose ultrafiltration membrane were characterized using CMC 535 TM and were responsive to polysorbate 80 but not to BigCHAP. The results are as Figure 10As shown. As shown, both 100 kDa and 50 kDa molecular weight cut-off (MWC) regenerated cellulose ultrafiltration membranes allow mixed micelles to pass through, while when 30 kDa and 10 kDa MWC ultrafiltration membranes are used, the retentate shows an increase in the amount of polysorbate 80.
[0051] It should be understood that while regenerated cellulose filters are commonly used due to their high porosity and relatively low non-specific binding, other materials used in ultrafiltration membranes (such as polyethersulfone (PES)) are manufactured by different methods and may exhibit different properties in terms of mixed micelle exclusion. Examples of the results of studies with PES ultrafiltration membranes of different MWC are shown as Figure 11 shown. As shown, PES membranes with 100 kDa and 50 kDa MWC show significantly greater mixed micelle retention than regenerated cellulose membranes of similar nominal molecular weight cut-off.
[0052] Figure 12 Shows the effect of different ultrafiltration membrane compositions on the recovery of BoNT / A from medium containing polysorbate 80, using BigCHAP as the second detergent. BoLISA TM assay is an immunological assay that is not significantly affected by residual polysorbate 80 and was used in these studies to quantify BoNT / A. As shown, the recovery of BoNT / A from regenerated cellulose and PES ultrafiltration membranes is similar, with a slightly lower recovery for PES.
[0053] To reduce non-specific binding to ultrafiltration membranes and other surfaces, non-specific binding sites available on these surfaces are typically blocked using proteins such as casein, gelatin, ovalbumin, bovine serum albumin, human serum albumin, non-specific immunoglobulins, etc. Such surfaces can be pre-blocked by protein treatment before exposure to the sample to be processed, or blocked during processing by adding protein (usually a small volume concentrated stock solution) to the sample to be processed, or both. However, such blocking proteins may have a negative impact on downstream processes such as characterization using in vitro methods (such as BoLISA TM and BoTest TM ) or cell-based assays (such as BoCell TM ).
[0054] Figure 13 Shows the effect of pre-blocking treatment of the ultrafiltration membrane used in the treatment of mixed micelles with human serum albumin (2% w / v, overnight) and the addition of 0.1% w / v human serum albumin to the processed sample for ultrafiltration membrane binding. The first detergent content of the sample was characterized using CMC 535 TM assay, and BoLISA TM and BoTest TMCharacterize the recovery of sample BoNT / A. As shown, adding human serum albumin to the sample and then treating it with an ultrafiltration membrane for pre-blocking had little effect on detergent removal, and slightly increased the recovery of BoNT / A when characterized by in vitro test methods such as BoLISA TM and BoTest TM .
[0055] Figure 14 Shows the effect of adding human serum albumin to a sample whose BoNT / A content is characterized. As shown, adding human serum albumin to a sample treated by the mixed micelle method and pre-blocking with an ultrafiltration membrane had a negative impact on the health of the cells used in the BoCell TM assay. Therefore, the effect of the blocking protein added to the sample treated by the mixed micelle separation method on the downstream process is process-dependent. TM
[0056] Those skilled in the art should be aware that, without departing from the inventive concept of the present application, more changes can be made in addition to the changes already described. Therefore, the subject matter of the present invention is not limited within the spirit of the appended claims. In addition, when interpreting the specification and claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components or steps in a non-exclusive manner, indicating that the recited elements, components or steps may exist, be used or combined with other elements, components or steps not expressly recited. If the specification or claims refer to at least one selected from the group consisting of A, B, C... and N, it should be interpreted as only requiring one element of the group, rather than A and N or B and N, etc.
Claims
1. A method for removing a first surfactant or detergent from a protein-containing solution, the method comprising: adding a second surfactant or detergent to the solution at a concentration at least equal to the critical micelle concentration of the second surfactant or detergent; providing sufficient time to allow the formation of a suspension of mixed micelles containing the first surfactant or detergent and the second surfactant or detergent; and separating the protein from the mixed micelle suspension by a size-based separation method.
2. The method according to claim 1, wherein the size-based separation method is ultrafiltration.
3. The method according to claim 2, comprising the step of blocking the ultrafiltration membrane used in the ultrafiltration method.
4. The method according to claim 1, wherein the size-based separation method is gel filtration.
5. The method according to claim 4, comprising the step of blocking the gel filtration medium used in the gel filtration method.
6. The method according to any one of claims 1 to 5, comprising collecting an analytical fraction containing the protein from the size-based separation method.
7. The method according to claim 6, comprising characterizing the analytical fraction by a cell-based assay.
8. The method according to any one of claims 1 to 7, wherein the first surfactant or detergent is selected from polysorbate 20, polysorbate 80, and Triton X-100.
9. The method according to any one of claims 1 to 8, wherein the second surfactant or detergent is selected from galactoside detergents, glucamide detergents, cholamide detergents, and sulfobetaine detergents.
10. The method according to claim 9, wherein the galactoside detergent is octyl-β-galactoside.
11. The method according to claim 9, wherein the glucamide detergents are selected from MEGA 8, MEGA 9, and MEGA10.
12. The method according to claim 9, wherein the cholamide detergents are selected from CHAPS, CHAPSO, and BIGCHAPS.
13. The method according to claim 9, wherein the sulfobetaine detergent is sulfobetaine 3-10.
14. The method according to any one of claims 1 to 13, wherein the protein is botulinum neurotoxin.
15. The method according to claim 14, wherein the botulinum neurotoxin is botulinum neurotoxin type A.