55 results about "Mixed-mode chromatography" patented technology

The present invention provides a manufacturing method for polypeptides that are produced in insect cells using a baculoviral expression system. In one example, the insect cell culture is supplemented with a lipid mixture immediately prior to infection (e.g., one hour prior to infection). The polypeptides are isolated from the insect cell culture using a method that employs anion exchange or mixed-mode chromatography early in the purification process. This process step is useful to remove insect-cell derived endoglycanases and proteases and thus reduces the loss of desired polypeptide due to enzymatic degradation. In another example, mixed-mode chromatography is combined with dye-ligand affinity chromatography in a continuous-flow manner to allow for rapid processing of the insect-cell culture liquid and capture of the polypeptide. In yet another example, a polypeptide is isolated from an insect cell culture liquid using a process that combines hollow fiber filtration, mixed-mode chromatography and dye-ligand affinity in a single unit operation producing a polypeptide solution that is essentially free of endoglycanase and proteolytic activities. In a further example, the isolated polypeptides are glycopeptides having an insect specific glycosylation pattern, which are optionally conjugated to a modifying group, such as a polymer (e.g., PEG) using a glycosyltransferase and a modified nucleotide sugar.

Use of mixed mode chromatography for purification of an antibody from an antibody mixture, for example) a Pichia pastoris fermentation mixture containing impurities such as host cell proteins and DNA is described Mixed mode chromatography is used instead of protein A chromatography and presents certain advantages over protein A chromatography Furthermore, the integration of such a method into a multi-step procedure with other fractionation methods for purification of antibodies suitable for in vivo applications is provided.

This invention relates to the use of mixed mode chromatography for purification of a protein from a mixture containing other materials, including fragmented or aggregated antibodies, host cell proteins, DNA, endotoxin, and/or virus. This invention further relates to the integration of such a method into a multi-step procedure with other fractionation methods for purification of antibodies or other proteins suitable for in vivo applications.

Proteins are purified by a mixed-mode chromatography system formed by attaching a ligand with cation exchange and hydrophobic functionalities to a large-pore support matrix, the only linkage between the ligand and the support matrix being a chain having a backbone of no more than three atoms between the hydrophobic group and the support matrix.

A mixed mode chromatography method for separating correctly folded from incorrectly folded conformations of a given protein is provided. The method is highly effective in separating correctly folded etanercept from incorrectly folded etanercept and aggregates in commercially attractive yields capable of affording etanercept preparations having very high purity in terms of correctly folded etanercept versus incorrectly folded etanercept. The invention is further directed to protein preparations and formulations comprising correctly folded proteins obtained using the present methods, and methods of treatment using the high purity preparations obtained from the mixed mode method.

The invention relates to a preparation method and applications of a phenyl boronic acid modified silica gel functional chromatographic filler, wherein Silica Gel is a silica gel, R1 is a phenyl boronic acid derivative, R2 is vinyl or mercapto, R3 is a co-modified modifying group, n is 0-4, and m is 0-3. The present invention provides the preparation method of the chromatographic filler, wherein vinyl or mercapto silane is bonded on the surface of a silica gel by using a modifying reagent such as a silylating agent to obtain an alkenyl or mercapto silica gel, and then the alkenyl or mercapto silica gel and a phenyl boronic acid derivative having mercapto or alkenyl are subjected to a mercapto-olefin click reaction to obtain the phenyl boronic acid modified silica gel functional chromatographic filler. According to the present invention, the preparation method has characteristics of high yield, good repeatability, mild reaction condition, and the like; and the prepared phenyl boronic acid modified silica gel functional chromatographic filler has characteristics of novel structure, a certain hydrophilicity, a certain hydrophobicity and a certain charge responsiveness, can be used as completely-new mixed mode chromatographic filler and the stationary phase, and can be widely used in the separation of various samples.

The invention discloses a strong cation exchange/hydrophobic mixed-mode chromatograph stationary phase, wherein X is -OCH3 or -OCH2CH3; R is shown in the specification, wherein n is equal to 1-5, or R is shown in the specification, wherein n is equal to 1-6; or R is PEG (Polyethylene Glycol) 200-1000. A preparation method comprises the following steps of: bonding cystine on the activated silica gel surface with hydroxyl groups by a silane coupling agent, then using DTT to open cystine disulfide bonds on the silica gel surface and form sulfydryl silica gel, then using H2O2 to oxidize the sulfydryl groups into sulfonic acid groups and form silica-gel derivatives bonded with the sulfydryl groups, and finally reacting with fatty alcohol (or aromatic alcohol or PEG and the like) to obtain the hydrophobic/strong cation exchange chromatograph stationary phase. The stationary phase can realize effective separation of proteins under the hydrophobic mode and the strong cation exchange mode, and one chromatographic column filled with a double-function separating medium can replace two common strong cation exchange/hydrophobic chromatographic columns to carry out separation and purification on the proteins.

The invention discloses a new method for separating out and purifying high-purity lysozyme from egg white. The method includes the steps that firstly, the surface of a silica gel matrix is decorated with tryptophan molecules serving as ligand of filler, and novel high performance hydrophobic interaction chromatography (HPHIC) filler regulated and controlled by static groups is synthesized; secondly, a chromatographic column which the HPHIC filler is filled with has the reserved characteristics of mixed mode chromatography, and has high selectivity and load capacity for natural protein separation; thirdly, the filler is applied to separation of an egg white sample, and high-purity and high-activity protein lysozyme with the purity of 99% and the quality recycling rate of 97.8% can be obtained. A salt-water elution system is adopted for the separation and purification process, and the new method is safe, environmentally friendly and beneficial for preparing lysozyme on a widened scale.

The present invention provides a method of purifying multimeric von Willebrand Factor (VWF) from a solution comprising multimeric VWF and contaminants. The method comprises passing the solution through a chromatography column comprising beads of a mixed mode chromatography resin coated with a size-exclusion inactive shell and collecting the multimeric VWF which passes through the column without binding to the resin.

The invention describes a method of purifying polysaccharide protein conjugates using mixed mode chromatography. The method involves contacting a crude polysaccharide protein conjugate with a mixed mode resin comprising an inert porous shell and an activated core under conditions of low conductivity that allow binding of the contaminants and collecting the unbound polysaccharide protein conjugate in a flowthrough.

The invention provides a separation method of proteins. A reversed phase/ion exchange mixed mode chromatography filler adopts a polar copolymerization bonding method and is adopted in the separation process of the separation method, a non-polar group and a polar group are simultaneously bonded on the silica gel surface, a separation mode is reversed phase/electrostatic repulsion interaction, and the separation method uses a reversed phase separation system; the reversed phase/electrostatic repulsion interaction means that 1) a non-polar group on the fixed phase surface provides reversed phasehydrophobic interaction; 2) a pH value of eluent is regulated from acid to neutral, so that a polar group on the fixed phase surface is positively charged; the pH value of the eluent is set to be lessthan an isoelectric point of each of the proteins required to be separated, so that the protein is positively charged; the positively-charged protein and the positively-charged polar group of a fixedphase filler generate electrostatic repulsive interaction, and the various proteins are separated due to different repulsive forces. The separation method provided by the invention has the characteristics of stability, high efficiency and unique selectivity.

The invention relates to a reversed phase/ion exchange mixed mode chromatography stationary phase, a preparation method and an application. The reversed phase/ion exchange mixed mode chromatography stationary phase is called as an octadecyl/sulfonic acid group mixed chromatography stationary phase, and consists of the octadecyl and the sulfonic acid group bonded to the surface of porous silica gel. The preparation method is as follows: bonding gamma-(2,3-epoxypropoxy)propyltrimethoxysilane KH-560 to the surface of the porous silica gel particle by the chemical vapor deposition method, and respectively carrying out a reaction of KH-560 as a linker arm with sodium bisulfite and octadecyl chloride to prepare the octadecyl/sulfonic acid group mixed chromatography stationary phase.

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.