40 results about "Mixed-mode chromatography" patented technology

The invention discloses a zwitter-ion mixed-mode chromatographic stationary phase as well as a preparation method and application thereof. The polymer is mainly composed of a bottom tertiary amine (alkaline) group and a top negative ion (acidic) group, and hydrophobic groups are distributed in the polymer. Different groups can be modified on the surface of the silica gel through a direct bonding or indirect assembling method, and the process is simple, controllable and repeatable. The zwitter-ion stationary phase is novel in composition, stable in structure and adjustable in surface property, has zwitter-ion polar groups and non-polar groups such as alkyl / aryl, can provide multiple interactions at the same time, and is suitable for separating different types of substances in multiple chromatographic separation modes such as hydrophilicity, reverse phase and ion exchange.

The invention discloses an efficient purification process of a bispecific antibody and a special chromatography system thereof. The efficient purification process of the bispecific antibody comprises the following steps: pretreatment: performing 0.22 mu m microfiltration on a cell culture supernatant to remove cell debris, and performing deep filtration to reduce turbidity to be less than or equal to 1NTU so as to reduce subsequent chromatography medium pollution; carrying out improved Protein A affinity chromatography, namely carrying out affinity chromatography by adopting an agarose medium modified by a recombinant Protein A ligand, taking 20mM Tris-HCl as a loading buffer solution, and taking the pH value of the loading buffer solution as 7.4, taking 100mM citric acid as an elution buffer solution, and taking the pH value of the elution buffer solution as 3.2-3.5; performing mixed mode chromatography: adopting a composite mode medium containing a benzamidine functional group, taking 50mM sodium phosphate as a buffer solution, adjusting the pH value to 6.0-6.5, adjusting the NaCl concentration to 0.1-0.3 M, and specifically removing a double-antibody polymer and a mismatched isomer by utilizing a hydrophobic effect and an ion exchange synergistic effect; and performing nanoscale filtration and terminal treatment: removing viruses by adopting a virus filtration membrane with the aperture of 30nm, and performing 0.22 mu m sterile filtration to obtain a final product. According to the process, the purification efficiency, the yield and the product purity are remarkably improved, and the process is suitable for efficient purification of the bispecific antibody.

The present invention relates to a method for purifying small extracellular vesicles by cation exchange mixed-mode chromatography.

The present invention relates to a method for purifying botulinum toxin (BTX). More specifically, the method is performed in the order of purification steps using cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), and mixed mode chromatography (cation exchange (phosphate) and affinity (calcium)), the method further comprising the step of treating trypsin to activate the botulinum toxin. Therefore, the method can purify botulinum toxin having excellent purity and activity and thus can be useful in producing botulinum toxin.

A mixed-mode chromatography method for the determination of phosphorylated sugars in a sample is provided. The mixed-mode chromatography method includes obtaining a sample comprising at least one phosphorylated sugar. The sample is introduced onto a chromatography system. The chromatography system includes a column having a stationary phase material contained inside the column. The stationary phase material has a surface comprising a hydrophobic surface group and at least one ionizable modifier. The sample with a mobile phase eluent is flowed through the column, where the at least one phosphorylated sugar is substantially resolved and retained within seven minutes. The mobile phase eluent includes water with an additive and acetonitrile with the additive. The mobile phase eluent has a pH less than 6. The at least one phosphorylated sugar is detected using a detector.

Disclosed herein is a method of purifying a protein, in particular a heme-binding protein, comprising passing a solution comprising a heme-binding protein and other proteins through a mixed-mode cation exchange chromatography resin under conditions that promote selective binding of the heme-binding protein to the resin, and eluting the bound heme binding protein from the resin. The disclosure also extends to compositions comprising the purified heme-binding proteins and uses thereof.

Chromatography resins having anionic exchange-hydrophobic mixed mode ligands and methods of using such resins are provided.

The present disclosure provides a stable protein composition containing a surfactant and having less than 400 subvisible particles of 10 microns or greater diameter per container, or less than 10,000 subvisible particles of 2 microns or greater per container. A method of manufacturing such a stable protein composition is disclosed, which includes a unit of operation that removes or decreases an esterase activity that degrades the surfactant. The unit of operation may be hydrophobic interaction chromatography or filtration, mixed mode chromatography, or the like.

The present invention relates to the field of chromatographic sample separation including liquid chromatography and solid phase extraction and in particular it relates to materials and the synthesis of materials for use as stationary phases in chromatographic sample separation. The invention also relates to the use of this material, in particular in the separation of hydrophilic and hydrophobic peptides, non-glycosylated and N-linked glycosylated peptides, deamidated and oxidized peptides. The invention also relates to a chromatographic column and a solid phase extraction column containing the material as a stationary phase.

The present disclosure provides a method for large-scale manufacturing of fXa-derived proteins to obtain a high yield of a high-purity protein product. The method may include adding a detergent to a sample containing a polynucleotide construct encoding the protein, and purifying the protein by affinity chromatography, ion exchange and mixed mode chromatography and hydrophobic interaction based on soybean trypsin inhibitor (STI).

The present invention relates to a method for purifying adeno-associated virus particles by cation exchange mixed-mode chromatography.

The present invention relates to methods for purifying small extracellular versicles by cation exchange mixed mode chromatography.

Chromatography resins having anionic exchange-hydrophobic mixed mode ligands, that are useful for purifying target biomolecules using anionic exchange (i.e., where the ligand is positively charged) and hydrophobic mixed mode chromatography. The chromatography resins allow for efficient purification of target biomolecules (e.g., recombinant proteins, antibodies, antibody-drug conjugates, or antibody derivatives including, but not limited to, antibody fragments and antibody fusions) from a sample, and have been found to be useful in purifying monomeric target biomolecules from aggregate target biomolecules. In an embodiment, the chromatography resins are useful for separating antibodies from one or more components (e.g., contaminants) in the sample.

Chromatography resins having anionic exchange-hydrophobic mixed mode ligands and methods of using such resins are provided.

The invention provides a method for removing an acidic isomer in a monoclonal antibody, and belongs to the technical field of antibody purification. According to the present invention, the monoclonal antibody solution is purified by using the affinity chromatography and mixed mode chromatography combined method, such that the acid isomer in the monoclonal antibody can be efficiently and stably removed, the loss of the target monoclonal antibody is reduced, and the product yield and the product purity are improved. Through detection, the content of the acid isomer in the monoclonal antibody can be reduced by more than 6% by adopting the method provided by the invention. In addition, the method is relatively simple and convenient to operate, is easy for large-scale production, can meet the requirements of industrial production, and has a good application prospect.

Chromatography resins having anionic exchange-hydrophobic mixed mode ligands and methods of using such resins are provided.

Preparation of Factor XA Derivatives.SOLUTION: The present disclosure provides methods for large-scale manufacturing of fXa derivative proteins to obtain high yields of high purity protein products. The method can comprise adding a detergent to a sample comprising a polynucleotide construct encoding a protein and purifying the protein via soybean trypsin inhibitor (STI) - based affinity chromatography, ion exchange and mixed mode chromatography, and hydrophobic interaction.SELECTED DRAWING: None

The invention relates to the technical field of high performance liquid chromatography stationary phases, and discloses a preparation method of a modified maleated rosin ester bonded silica gel mixed mode stationary phase and application of the modified maleated rosin ester bonded silica gel mixed mode stationary phase. Bonding on the surface of silica gel through a mercapto-alkene click reaction to prepare a mixed-mode chromatographic stationary phase; the rigid hydrophenanthrene cyclic ester provides hydrophobic interaction sites for the stationary phase, the tertiary amine and the hydroxyl group provide hydrophilic interaction sites for the stationary phase, the protonated tertiary amine provides ion exchange sites, and the mixed mode stationary phase has an RPLC / HILIC / IEC chromatographic mode. The preparation method of the stationary phase is simple, the requirement on an experimental device is low, the stability of a chromatographic column is good, and the stationary phase has good application potential. A chromatographic column prepared by the mixed-mode chromatographic stationary phase can quickly and efficiently separate hydrophobic, hydrophilic and organic acid solutes, and has a good separation effect on panax notoginseng saponins.

The invention discloses a small nucleic acid medicine purification process, and belongs to the technical field of nucleic acid purification. A small nucleic acid drug purification process comprises the following steps: S1, mixed mode chromatography: dissolving a small nucleic acid drug crude product in a buffer solution containing 20mM phosphate and 10% acetonitrile to obtain a solution A; loading the solution A to a mixed-mode chromatographic column with a mixed-mode chromatographic column medium comprising functionalized agarose gel, and carrying out gradient elution to remove impurities; eluting the target product with a buffer solution containing 2M NaCl and 10% acetonitrile; s2, size exclusion chromatography: diluting a small nucleic acid drug sample eluted by mixed mode chromatography with an equilibrium buffer solution, loading the sample into a size exclusion chromatography column, performing isocratic elution with the equilibrium buffer solution, monitoring an ultraviolet absorption signal at 260nm, collecting an elution peak, and freeze-drying to obtain a high-purity small nucleic acid drug; the functionalized agarose gel is agarose gel for simultaneously modifying a quaternary ammonium salt group and an alkyl group.

The invention relates to a preparation method of a hydrophilic interaction-anion exchange mixed mode chromatographic stationary phase, which comprises the following steps: by taking silica gel microspheres as a matrix, reacting silicon hydroxyl on the surface of the silica gel microspheres with a first modified compound, and bonding a layer of epoxy compound; then, by taking BF3 as a catalyst, carrying out ring-opening reaction on hydroxyl on the second modified compound and an epoxy group of the first modified compound under an anhydrous condition so as to graft the second modified compound onto the surface of the silicon ball; the second modification compound comprises one or more mixtures containing a plurality of hydroxyl groups or / and amino groups. The surface of the obtained stationary phase has a large number of hydroxyl groups and amino groups at the same time, and a mixed mode chromatographic separation performance of hydrophilic interaction chromatography and anion exchange chromatography can be provided, so that special separation selectivity is provided for separation of some hydrophilic compounds and ionic compounds.

The present invention relates to a polysorbate separation method, comprising a polysorbate-containing solution to be measured flowing through a mixed-mode chromatograph and a reversed-phase chromatograph which are coupled in series, the use of the separation method in measuring the polysorbate content of said polysorbate-containing solution, a related apparatus, and a method for measuring the polysorbate content by using the separation method.

An L-lysine-derived gel factor mixed-mode chromatography stationary phase, wherein the L-lysine-derived gel factor mixed-mode chromatography stationary phase is based on silica gel, and the bonding phase groups on the surface of the silica gel matrix include hydrophilic amide groups, hydrophobic tert-butyl groups and fluorene groups; the preparation method for preparing the stationary phase includes: synthesizing an L-lysine-derived gel factor; preparing amino silica gel; synthesizing an L-lysine-derived gel factor-modified mixed-mode chromatography stationary phase; the application of the stationary phase is for application in multi-chromatographic separation in the same column. The stationary phase synthesized in the present invention contains hydrophilic amide groups, hydrophobic tert-butyl groups, fluorene groups, etc., and can provide various interactions such as hydrophobic interaction, π-π stacking interaction, hydrogen bond interaction and hydrophilicity. The stationary phase can be used as a chromatographic column to realize the application of multi-chromatographic separation mode in the same column during the separation process, making up for the defects and deficiencies shown by a single chromatographic mode when separating complex samples.

The invention discloses a mixed mode chromatographic stationary phase based on cyclic polyamine and a preparation method thereof, N-monoalkylated piperazine is firstly obtained through a selective N-alkylation technology, then the N-monoalkylated piperazine is converted into silane, then the silane is immobilized on silica gel, and the novel mixed mode chromatographic stationary phase based on cyclic polyamine is prepared. Selective N-alkylation adopted in the method has reaction specificity, silane synthesis has diversity, and the obtained stationary phase has remarkable alkalinity, excellent hydrophilicity and stability, is suitable for analyzing high-activity electrophilic reagents, weakens the tendency that a silyl ether bond between a bonded phase and a silica gel substrate is damaged by a protonic solvent in a mobile phase, and improves the stability of the stationary phase. Loss of a chromatographic stationary phase is reduced, and the chromatographic retention capacity is improved.

The present invention relates to the field of chromatographic sample separation, including liquid chromatography and solid phase extraction, and in particular it relates to materials for use as stationary phases in chromatographic sample separation and to the synthesis of materials. The invention further relates to the use of materials in the separation of hydrophilic and hydrophobic peptides, non-glycosylated peptides and N-linked glycosylated peptides, deamidated peptides and oxidized peptides. The invention also relates to a chromatographic column and a solid phase extraction column containing the material as a stationary phase.

The present disclosure provides a stable protein composition containing a surfactant and having less than 400 subvisible particles of 10 microns or greater diameter per container, or less than 10,000 subvisible particles of 2 microns or greater per container. A method of manufacturing such a stable protein composition is disclosed, which includes a unit of operation that removes or decreases an esterase activity that degrades the surfactant. The unit of operation may be hydrophobic interaction chromatography or filtration, mixed mode chromatography, or the like.

Disclosed herein is a method for purifying proteins, particularly hemopexin, comprising passing a solution containing hemopexin and other proteins through a mixed-mode cation exchange chromatography resin under conditions that promote selective binding of hemopexin to the resin, and eluting the bound hemopexin from the resin. The disclosure also extends to compositions containing purified hemopexin and uses thereof.

The present invention provides methods of purifying multispecific antibodies. The methods comprise the sequential steps of performing a capture chromatography, a first mixed mode chromatography and a second mixed mode chromatography. In some aspects, the invention provides compositions of multispecific antibodies, which compositions have reduced levels of one or more product-specific impurities and / or process-specific impurities.

Herein is reported a method for the purification of an antibody directly captured from clarified cell culture supernatants using Streamline CST and / or Capto MMC, wherein especially product related (aggregates and fragments) and process related impurities (host cell protein, media components) could efficiently be removed, resulting in a preparation with a purity comparable to classical protein A affinity chromatography.

The invention discloses a method for detecting nicergoline related substances. The method comprises the following steps: preparing a test solution; preparing a contrast solution; and respectively injecting the test solution and the contrast solution into a high performance liquid chromatograph, and simultaneously detecting various related substances of nicergoline under the same chromatographic condition by using a mixed-mode chromatographic column through a gradient elution procedure. By adopting the strong cation exchange and reversed-phase C18 mixed filler chromatographic column and combining with an optimized ion pair reagent buffer system and a gradient elution procedure, all key impurities in nicergoline can be detected at one time by adopting a set of liquid chromatography system, so that the detection efficiency is improved by about 50%, the operation process is greatly simplified, and the detection cost is reduced. Solvent consumption and personal errors are reduced; the separation degree of each impurity peak and the main peak is greater than 2.0, the peak shape is symmetrical, the system adaptability is good, the method reproducibility is excellent, and a more efficient, comprehensive and reliable analysis means is provided for the quality control of nicergoline.

The application provides a method for determining three items of urinary calculi by mixed mode chromatography-tandem mass spectrometry without derivation, which comprises the following steps: mixing a sample to be measured with a dilution solution, centrifuging, then performing liquid chromatography-mass spectrometry detection, and calculating the content of the three items of urinary calculi in the sample to be measured according to the detection result. The method provided by the application relies on the unique multiple physical and chemical synergistic mechanism of the mixed mode stationary phase and the precise pH elution kinetics regulation, completely solves the stereoisomer interference, significantly improves the accuracy of clinical quantification, effectively avoids the "dead volume" flushing, strongly overcomes the matrix inhibition effect of oxalic acid, resolves the physical and chemical conflicts, takes into account the perfect peak shape of simultaneous detection of special amino acids and organic acids, abandons high-pollution reagents, constructs a "green flow path" for long-life operation of mass spectrometry, greatly simplifies the pretreatment and rapidly elutes the chromatography, and perfectly adapts to the high-throughput automatic pipeline.