CN101444757B - Porous adsorbing medium, the adsorbent equipment containing this medium and tomographic system - Google Patents
Porous adsorbing medium, the adsorbent equipment containing this medium and tomographic system Download PDFInfo
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- CN101444757B CN101444757B CN200810171406.0A CN200810171406A CN101444757B CN 101444757 B CN101444757 B CN 101444757B CN 200810171406 A CN200810171406 A CN 200810171406A CN 101444757 B CN101444757 B CN 101444757B
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J41/00—Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
- B01J41/20—Anion exchangers for chromatographic processes
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- B01J47/00—Ion-exchange processes in general; Apparatus therefor
- B01J47/12—Ion-exchange processes in general; Apparatus therefor characterised by the use of ion-exchange material in the form of ribbons, filaments, fibres or sheets, e.g. membranes
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Abstract
Medium and device, such as, comprise the anion exchanger of this medium, and its medium is the film that surface is coated with polymer such as polyallylamine.The film obtained shows the combination stronger with protein impurities and better from biological sample, removes theme cell protein than the conventional such as trimethyl ammonium part of the part based on quaternary ammonium salt.Application also describes tomographic system and the method for monoclonal antibody purification, wherein Anion-adsorption device is placed on the downstream of affinity post (such as a-protein or Protein G affinity post), and choose any one kind of them or multiple burnishing device, such as cation exchange column.Cation exchanger pond (or affinity post exchanges pond, does not wherein use cation exchanger) is few or do not have the conductibility of diluting reduction sample to be necessary.This absorber even also has the good function combined the host cell proteins matter in biological sample and other impurity brute force under high conduction and pH.
Description
The application's priority is the U.S. provisional application Ser no.61/070708 that the U.S. provisional application Ser no.60/964653 and 2008 submitted on August 14th, 2007 submits to 25, on March, and it is incorporated to the application by reference to document.
background of invention
The application relates to the anion-exchange chromatography medium based on polymerism primary amine, comprises the Anion-adsorption device of that medium, and comprises the tomographic system of this absorber.Absorb the body absorption material referred to by penetrating into sorbing material.Absorption refers to molecule and moves to mutually the surface of adsorbing medium from body.Suction-operated is the general name comprising absorption and absorb.Same, sorbing material here or adsorbent equipment represent absorber, refer to material or the device of absorption or absorption or absorption and absorption.This medium is applied as especially for the porous membrane absorber of circulating module and more particularly not containing the module of independent mistress.
Strong anion exchanger, such as those are based on the anion exchanger of quaternary ammonium salt, downstream process is used for catch electronegative large-scale impurity as polishing medium, such as endotoxin, virus, nucleic acid and host cell proteins matter (HCP), they are present in the solution of fluid such as biofluid, particularly artificial creature's therapeutic agent.Traditionally, anion exchanger provides with the form of bead and uses, such as, from the QSepharose that GEHealthcareBio-sciencesAB is commercially available
.But the restriction of the output of bead based system needs the pillar of large volume effectively to catch impurity.
In the chromatography of bead base, the surface area that major part can be used for adsorbing is in the inside of bead.Therefore, due to mass transfer rate typically by hole spread control, separation process is in fact very slow.Minimize to make this diffusion resistant and along with maximized dynamic binding capacity, the bead of minor diameter can be used.But the use of minor diameter bead makes post drops while improve price.Therefore, the optimization that preparative chromatography is separated is usually included between efficiency/dynamic capacity (small-sized bead easily occurs) and post drops (large-scale bead easily occurs) compromise.
On the contrary, film basic unit analysis system (being also called film absorber) has the part be directly connected with convection current membrane pores, thereby eliminates the impact of internal void diffusion on mass transfer.In addition, the use with the microporous barrier matrix of fine and close pore size distribution be combined with effective flow distributor can make axial dispersion minimize, and provides the homogeneous utilization of all avtive spots.Therefore, the mass transfer rate of film absorber medium can an order of magnitude higher than the bead base chromatography media of standard, and this just allow for high efficiency and high-throughput isolation.Because single or even stacking film are very thin compared with the pillar with bead base media accumulation, find that the Pressure Drop along chromatography bed reduces, thus flow rate and productivity ratio are increased.By using the film with enough internal surface areas, production has very large diameter and reaches necessary binding capacity with the device of length ratio (d/h) profile.
Design suitable film absorber and there is the bead base resin good 10-100 more prefabricated than standard chromatography efficiency doubly.Therefore, in order to obtain the separation of phase same level on film absorber, can use and make a call to 10 following height of bed of folding.For film absorber, compared with the bead based system of the 10-30cm height of bed, the bed of 1-5mm is long is standard.Film absorber due to large volume requires extreme cylindricality ratio, and the design of device is critical.In order to keep the advantage of the inner behavior be associated with film absorber, require that suitable import and outlet distributor are with efficiently and effectively utilize the membrane volume of acquisition.What film adsorber technology was desirable is applicable to this application.But present film absorber has various shortcoming, comprises low bond strength, removing the difficulty shown in virus, endotoxin and nucleic acid.
When the latter flows through its hole, highly porous, the interconnective film absorber of medium has the ability of removing (absorption and/or absorb) some solution components.This character and it of film absorber show good ability and depend on the pore structure of medium (skeleton) and be exposed to the character on the surface in solution in required application.Typically, first by water insoluble or in water swelling polymer form porous medium, and it has acceptable engineering properties.The porous diaphragm that porous medium is prepared preferably by the known phase disengagement method of prior art.See, such as ZemanLJ, ZydneyAL,
microfiltrationandUltrafiltration:PrinciplesandApplicati ons,newYork:MarcelDekker, 1996.Doughnut and tubular film are also acceptable skeletons.Usually require that the outside of the cellular structure that separate processing step is formed with modification or front face surface and internal void surface are to give the characterization of adsorption of its necessity.Because membrane structure is often formed by hydrophobic polymer, another object of surface modification step be to make surface become hydrophilic or water wettable.
Here there is the outside of a large amount of Modified Membrane or the method on front face surface and internal void surface.Those those skilled in the art will be easy to recognize representative method, comprise absorption, plasma oxidation, original position radical polymerization, grafting and coating.The great majority of these methods all cause coordinating on the surface of the film the structure of similar individual layer, and this obtains the target making it hydrophilic most of time, but but can not give its acceptable characterization of adsorption, such as, for the high power capacity of absorbate.This capacity definition is the amount (weight) of the absorbate that medium volume that can be given retains.Since absorption occurs in the surface of film, this capacity will be subject to the restriction of film surface area.Due to their characteristic, microporous barrier has lower surface area compared with chromatography beads.Increase the method for its surface area for reducing a pore-size, this significantly causes losing a large amount of flux.Such as, not considering the type of surface interaction, is about 20mg/ml in the maximum (individual layer) of the upper protein adsorption of 0.65 μm of polyethylene film (EntegrisCorp, BillericaMA).This is far less than such as agarose chromatography beads, and its typical capacity is about 80mg/ml.
The type promoting the surface interaction of absorption is defined by the application-specific of wherein used given film absorber product.Now, also exist from the solution of monoclonal antibody (MABs) except virus removal, nucleic acid, endotoxin and the high power capacity of host cell proteins matter (HCPs), the needs of high affinity absorber.These impurity tend to have the isoelectric point lower than MABs, this just to mean under a certain pH value they will be electronegative and MAB to fall be electropositive.Requirement anion exchanger, namely carries positive charge and attracts the medium of anion to remove these impurity.Many chemical compositions, in aqueous all with positive charge, comprise primary, secondary and tertiary amine and quaternary ammonium salt.These amine pH value lower than 11 time be all electropositive, and quaternary ammonium salt under all pH value all with positive charge, therefore these groups are called as weak or strong anion exchanger usually respectively.
Anion exchanger has multiple attraction and retains the electropositive binding site of plurality of impurities and pollutant.Can the amount of impurity of potential removing be the function of these binding site concentration on film, and determine its bond strength to different impurities due to the chemical characteristic (and concentration of these parts) of part.The combination of high strength is such as, to raising impurity, host cell proteins matter, the key contribution of removal.Bond strength (SB) relates to the solution ion strength required for impurity of elution of bound.The SB (weighing with conduction unit, mS/cm) of film absorber measures as follows.First, the solution of a small amount of absorbate is made to be combined on film absorber to make absorbate by film absorber.The second, with the inorganic salts increasing gradient, such as sodium chloride, wash-out film absorber.Record needs the minimum conductibility of the elute soln washed away by absorbate and is defined as the SB of film absorber.By increasing the bond strength of absorber, can make that electonegative impurity is irreversible to be combined on film absorber, improve effect of removal thus significantly.This is particular importance for the weak binding population removing host cell proteins matter from antibody stream.
Conventional circulation anion exchanger typically attracts containing being responsible for and in conjunction with electonegative impurity, but repelling the quaternary ammonium salt part of electropositive product molecule.General knowledge requires that the anion exchanger that namely strong anion exchanger all has positive charge in all pH value is necessary in order to the interaction by electric charge is combined with impurity strongly.And present invention demonstrates that other mode.The present invention finds polymerism primary amine, preferably there is the aliphatic polymer of covalently bound primary amine on main polymer chain, more preferably have by the covalently bound primary amine on main polymer chain of at least one aliphatic group, be preferably methylene group, stronger be combined with the material of removing electronegative impurity and be therefore preferably for forming the material category of adsorptivity hydrogel on the surface of film absorber.
Monoclonal antibody continues to have importance as treatment and diagnostic reagent.Method for the screening hybrid cell storehouse of candidate mABs is not only consuming time but also take a lot of work.Once the hybrid cell chain indicating suitable mABs is set up, purification process just must be developed to prepare enough mAB for characterizing further.Traditional method for purifying comprises use a-protein or Protein G affinity chromatography.Make the antibody desalination of purifying and in biological buffer, use dialysis to exchange.If parallel the carrying out of multiple mABs is evaluated, whole processes typically needs to complete over these days and particularly troublesome.
U.S. patent No.6090288 teaches the preparation of the amino group containing the chromatography media for polypeptide and separate nucleic acid.There is disclosed compared with strong anion exchange part, exchanging elute protein part from weak anionic needs higher ionic strength.The important architectural feature of disclosed separating medium is " in distance, ammonia nitrogen 2 to 3 atom places exist oh group or primary, secondary or tertiary amine groups ".Here, such as, we point out pure polyallylamine polymers loose cross-linked coating and without any need for promoting the extra group with protein more high strength bond.
U.S. patent No.5304638 teaches the method for separating protein using and comprise the water-insoluble matrix being loaded with numerous polyamine groups.Where one embodiment shows the agarose chromatography gel preparing polyallylamine modification.But 5304638 inventions are not recognized and are used primary amine and the importance using secondary amine to compare with tertiary amine.Not to control coating layer thickness with optimization suction-operated relevant or with for the relevant effort of stable cross-linked coating or description.They emphasize preferred nitrogen-atoms between carbon number be no more than 3.They describe the empirical function Q of a structure based on polyamine groups and pH meter calculation, and it has the value of preferred at least 1.5.In polyallylamine, between adjacent nearest nitrogen-atoms, have 5 carbon atoms, and the Q function for it should close to 0.1.
U.S. patent No.5547576 teaches that preparation is a kind of to be had fixing polyamine coating and may be used for from the aqueous solution except the porous membrane of virus removal.The preparation of coating comprises first at the surface grafting free radical of film, and then makes free radical and polyamino compound react.Due to the complexity of inherence, graft modification is often unpractical: they are responsive to the special matrix of free radical grafting, and to implement in a manufacturing environment be expensive.This method is also subject to the infringement of the structural disadvantage discussed in 5304638.
These three patents all, 6090288,5304638 and 5547576 all do not recognize the thickness of control polyamine coating or the importance of the degree of cross linking of polymer in that coating.All they all depend on the chemical reaction being fixed on the reactive group on surface containing the compound of amine and covalency, by grafting or directly react.Last in these processes any, is subject in fact the restriction of single-layer type containing amine coating.The high adsorption capacity of these films only can obtain, as the situation with agarose chromatography beads by increasing surface area.Present invention teaches by setting up relatively thick loose crosslinked polymerism layer on film surface and obtain high adsorption capacity, a kind of diverse ways of fundamentally instructing with all that prior art.
Therefore, expect the circulation anion exchanger providing medium He comprise this medium, it provides and to be combined with the brute force of virus and can not to be subject to the infringement of prior art shortcoming.When placing it in the downstream of affinity chromatographic column, optionally in the race that one or more cation exchange column carries out after step time, this interchanger is used in particular for the monoclonal antibody using tomographic system purifying from cell culture medium.
summary of the invention
Instant invention overcomes the problems of the prior art, the invention provides medium and device, such as, comprise the anion exchanger of this medium, its medium is coated with polymer for having surface, such as polyallylamine (polyallylamine), film.The film obtained is beat all provide than routine based on quaternary ammonium salt, the ability comprising the stronger conjugated protein impurity of trimethyl ammonium part and the more outstanding ability removing host cell proteins matter from biological sample.
The invention describes a kind of method significantly improving the adsorption capacity of microporous barrier.Replace the surface of Modified Membrane in similar mono-layer fashion, be coated with whole outside and internal void surface with loose crosslinked hydrogel, wet (swelling) thickness of this hydrogel is about 50-100nm, and this is enough to hold several layers protein molecule.Therefore, the adsorption capacity of microporous barrier is increased to 80-100mg/ml from about 20mg/ml.The interactional type promoting absorption defines by using the application-specific of given film absorber product.Present existence removes virus removal, nucleic acid, endotoxin and the high power capacity of host cell proteins matter (HCPs), the needs of high affinity absorber from the solution of monoclonal antibody (MABs).Many chemical compositions, in aqueous with positive charge, comprise primary, secondary and tertiary amine, and quaternary ammonium salt.Amine pH value lower than 11 time be electropositive, and ammonium salt under all pH value all with positive charge, therefore these groups are called weak or SAX.The present invention finds polymerism primary amine, preferably there is the aliphatic polymer of covalently bound primary amine on main polymer chain, more preferably it has by least one aliphatic group, preferably methylene group is covalently bound on the main chain of polymer, strongly be combined with electronegative impurity, and be therefore the material category being preferably used for being formed on the surface of film absorber adsorptivity hydrogel.
Invention further describes a kind of tomographic system and the method for monoclonal antibody purification, wherein anion exchange absorbing device is placed on affinity post (such as a-protein or Protein G affinity post) and chooses any one kind of them or several burnishing device such as cation exchange column or be the downstream of cation exchange column just.Consider the characteristic of described anion exchange absorbing device medium, few dilution or not dilute cation exchange pond (or not using the affinity post of cation exchanger to exchange pond) be necessary for reducing the conductivity of sample, this is because absorber medium of the present invention can run under higher ionic strength.Absorber even also has good strong binding function with the host cell proteins matter in biological sample and other electronegative impurity under conductivity is close to 15mS/cm and relative high ph-values.
As discussed before, because the convection current of the film absorber compared with the diverging flow of bead base absorber, present invention obtains relatively high device permeability and do not lose binding capacity.
summary of drawings
Figure 1A is the water flux of film absorber post and the curve map of BSA capacity that are cross-linked preparation with the PEG-DGE of PAH and 0.5% of 1mm;
Figure 1B is the water flux of film absorber post and the curve map of BSA capacity that PAH and the PEG-DGE with 9wt% of 1mm is cross-linked preparation;
Fig. 2 is the curve map of the BSA capacity of the film absorber of alkali-free form and sulfamic acid ionic species in accelerator card life test.
the detailed description of particular embodiment
The present invention relates to a kind of porous chromatography or the adsorbing medium with porous, the polymerism coating that porous is formed and self-sustained matrix, a kind of anion exchanger comprising this medium, comprises purification system and the purification process of this absorber.This medium is particularly suitable for from artificial creature's therapeutic agent such as monoclonal antibody, removing low-level impurity in the mode be present in downstream purification process that entirety is good forcefully.Typical impurity comprises DNA, endotoxin, HCP and virus.This medium has good effect under high salt concentration and high conductance (high affinity), also effectively can remove virus removal even under these conditions.Which give high binding capacity and do not damage the permeability of device.In fact, be determined by the character of coating prepared by method described herein, obtain and be greater than 5mg/ml, or be greater than 25mg/ml, or be greater than the binding capacity of nucleic acid of about 35-40mg/ml.Because the highly diluted sample be carried on absorber medium is no longer necessary, the amount of anion exchange absorbing device is far smaller than the amount of the bead based method compared.
Porous matrix has two surfaces relevant to the geometry of matrix or physical arrangement.Lamella has top and lower surface, or the first and second surfaces.These are commonly referred to " side ".In use, fluid will flow through opposite side (surface) from side (surface).For doughnut and tubular film, it has surface, inner side and outer side.Flowing is carried out from inner side to outside, or vice versa, and this depends on design and purposes.
The dimension of the thick layer (thickness) between two surfaces is porous.This porous region has the surface area relevant to hole.In order to prevent obscuring with " surface " " surface " or " surface area " or similar term, geometric jacquard patterning unit surface refers to as outside or front surface or side in the present invention.The surface area relevant to hole refers to inside or porous surface amasss.
Porous material is containing porose, and it be empty space, and makes the solid matrix of physical unit or the skeleton of material.Such as, in nonwoven web, the fiber of arbitrary orientation is made matrix and is given net with the form of himself.In polymer microporous film, the polymer that is separated provides matrix.Here, inventor discusses the surface of brushing or overwrite media.By this method, inventor is intended to brushing inner surface and outer surface, so that can not block hole completely, namely the structure of major part for convection current is remained, especially, for internal surface area, brushing or cover and mean brushing or cover matrix, and leave most hole and open.
Suction-operated refers to the body absorption material by penetrating into sorbing material.Suction-operated refers to molecule and moves to mutually the surface of adsorbing medium from body.Suction-operated is the general name comprising absorption and absorb.Same, sorbing material here or adsorbent equipment represent absorber, refer to material or the device of absorption or absorption or absorption and absorption.
Porous matrix act as the support frame for adsorptivity hydrogel.This matrix should submit to process and manufacture strong and complete device.Pore structure should provide uniform flow distribution, high flux and high surface.Matrix can be fiber, lamella such as textile fabric, adhesive-bonded fabric, paper, felt or film.Preferably, matrix is the lamella formed by fabric or adhesive-bonded fabric or film.
Fiber can be any length, diameter and can be hollow or solid-state.As matrix they and not together (although after application brushing, they can form single structure), but entity discrete separately.They can be line or the monofilament of the form such as uncertain length of continuous length, or prepare the shorter individual fibers of formation by chopping up fibrous material such as adhesive-bonded fabric or textile fabric, the fibre cutting of continuous length is become independent section, is formed by crystalline growth method or similar method.
Adhesive-bonded fabric directly adopts heat or chemistry to be wound around combine smooth, the porous sheet made by the fiber be separated by making fiber or filament.Typically, adhesive-bonded fabric producer provides the medium with 1 to 500 microns of mean flow pore gap (MFP) grades.For adhesive-bonded fabric, cellular structure is the fiber be wound around, and porous refers between fiber or among tortuous space.Preferred adhesive-bonded fabric is the FreudenbergNonwovensNA of Lowell, Mass., and its model is FO2463.
Textile fabric by meridional fibers and parallel fiber with the mode of rule or weave pattern some each other predetermined angle braiding make.Angle between typical parallel and warp is about 90 degree.Other normally used angle includes but not limited to 30,45,60 and 75 degree.The integrality of fabric is kept by the fibre machinery interlocking caused by fabrication processes.Surface smoothness and the stability of fabric (fabric meets the ability of complex surface), fabric control mainly through weave pattern, such as plain weave, twill, satin weave, basket shape fabric, petinet etc.In this case, the porosity of matrix is space between fabric and it seldom has tortuous character.
Matrix can also have multiple material to be formed, and comprises glass, plastics, pottery and metal.
Borosilicate glass is an example of suitable glass.It can form fiber or glassine paper.
Also the multiple pottery based on more conventional silicate chemistry or special chemistry such as yttrium, zirconium, titanium and analog and their blend can be used.They can form fiber, paper, felt, monolithic or film.
Metal is stainless steel, nickel, copper, iron or other magnetic metal and alloy such as, palladium, tungsten, platinum and analog may be prepared into various ways, comprise fiber, sintered sheets and structure, the stainless steel such as sintered or nickel filter, fabric sieve and non-woven paper, fabric and felt, such as stainless steel wool.
Preferred matrix is made up of that synthesize or natural polymeric material.Thermoplastic is the type of polymer for this purposes.Thermoplastic includes but not limited to polyolefin, such as polyethylene, comprise ultra-high molecular weight polyethylene, polypropylene, covering polyethylene/polypropylene fiber, PVDF, polysulfones, polyether sulfone, polyarylsufone, polyphenylsulphine, polyvinyl chloride, polyester is PETG, polybutylene terephthalate (PBT) and analog such as, polyamide, acrylate is polymethacrylates such as, the mixture of styrenic polymer and above-mentioned substance.Other synthetic material comprises cellulose, epoxides, carbamate and analog.
Suitable matrix comprises miillpore filter, and namely those have the filter membrane that about 0.1 arrives about 10 μm of pore-sizes.Host material can be hydrophilic or hydrophobic.The example of hydrophilic matrix material includes but not limited to polysaccharide and polyamide, and surface-treated hydrophilic porous property film, such as
(MilliporeCorporation, BillericaMA).The example of hydrophobic material includes but not limited to polyolefin, polyvinylidene fluoride, polytetrafluoroethylene (PTFE), polysulfones, Merlon, polyester, polyacrylate and polymethacrylates.Form cellular structure by those any methods known to those skilled in the art by host material, such as solution phase in version, temperature induced phase-separable, air curtain coating, vestige etches, and stretches, sintering, laser drill etc.Due to the character that the present invention is general, in fact the method for any available formation cellular structure is all applicable to the support frame for the preparation of film absorber.Have been found that the host material become by ultrahigh molecular weight polyethylene is useful, this is because its engineering properties, chemistry, corrosion and the associating of γ stability.
Coated polymeric forms adsorptivity hydrogel and retains the chemical group (conjugated group) of impurity with responsible attraction.Selectable, coated polymeric contains and is easy to modification and the chemical group being incorporated to conjugated group.Coating can penetrate biomolecule, therefore can capture protein and other impurity in the depths of coating, improves adsorption capacity.Preferred coated polymeric is polymerism primary amine.The example of suitable polymerism primary amine comprises polyallylamine, polyvinylamine, poly-butylamine, polylysine, they each other and with the copolymer of other polymer, and their corresponding protonated form.Find that the coating such as, be made up of polyallylamine (and/or its protonated form, nicotinic acid polyallylamine (PAH)) is useful especially.PAA is commercially available (NittoBoseki), and its number-average molecular weight is usually in the scope of 1000 to 150000, and all these all can be used for forming film absorber.PAA and PAH is easy to water-soluble.The pH value of the PAA aqueous solution is about 10-12, and that PAH is 3-5.PAA and PAH can replace use mutually, but must monitor the pH value of last solution, and if necessary, pH value is adjusted to be greater than 10 to such an extent as to the amino group of aprotic can be used for reacting with crosslinking agent.
Coating typically by least about 3% the forming of cumulative volume of brushing matrix, is preferably about 5% to about 10% of matrix cumulative volume.In certain embodiment, coating covers in matrix with homogeneous thickness substantially.The thickness range that dry coating is suitable is that about 10nm is to about 50nm.
Crosslinking agent and polymer reaction make the latter water insoluble and are therefore retained on the surface of support frame.Suitable crosslinking agent is two senses of reacting with coated polymeric or polyfunctional molecule, and may be dissolved in the solvent chosen, and it is preferably water.The most noticeable is epoxides with the chemical composition of the broad variety of primary amine reaction, chlorine, bromine and iodine alkane, carboxylic acid anhydrides and halide, aldehyde, alpha, beta-unsaturated esters, nitrile, acid amides and ketone.Preferred crosslinking agent is polyethyleneglycol diglycidylether (PEG-DGE).It is easy to water-soluble, provides quick and is effectively cross-linked, and hydrophilic, neutral, nontoxic and be easy to obtain.Amount for the crosslinking agent of coating solution is the mol ratio based on the reactive group on polymer and crosslinking agent.Preferred ratio, in the scope of about 10 to about 1000, is more preferably about 20 to about 200, most preferably is about 30 to about 100.More crosslinking agent will hinder the ability of swelling behavior and therefore will reduce adsorption capacity, and less crosslinking agent will cause incomplete crosslinked, namely remains some completely soluble polymer molecules.
Surfactant can be used for helping polymer solution is dispersed on all surfaces of supporting construction uniformly.Preferred surfactant be non-ionic, water miscible and alkalescence stable.Fluorine surfactant has the significant ability reducing water surface tension.These surfactants have E.I.duPontdeNemous and Company to take Zonyl as the material that trade name is sold, and specially suitablely have such as ZonylFSN and ZonylFSH.The kind of other acceptable surfactant is ethyoxyl octyl phenol, the material that DowChemicalCompany sells with trade name TritonX.Also other surfactant known by those those skilled in the art can be used.Concentration for the surfactant in coating solution is generally the surface tension of reduction solution to avoid the minimum of dewetting.After dewetting is defined as initial dispersion, liquid spontaneous one-tenth from the teeth outwards drips.During formation film absorber, dewetting is highly less desirable thing, and it sometimes causes non-wetted product and reduces adsorption capacity.The contact angle determination of the drop that the flat surfaces made with the material identical with porous matrix by measurement that the amount of required surfactant can be conventional is made.Powered Propulsion and receding contact angle are useful especially, and they are measured respectively as the liquid adding drop or fetch from drop.Dewetting can be avoided to the receding contact angle with 0 ° if regulated and controled by solution.
The a small amount of hydrophilic polymer being easy to be adsorbed on hydrophobic surface can optionally join in solution as dispersing aid.Polyvinyl alcohol is preferred polymer and can uses with the concentration within the scope of about 0.05wt.% to about 5wt.% of overall solution volume.
When the cellular structure supported can not be easy to soak with polymer solution, such as, when dewatering microporous film, wetting aid can be joined in solution.Wetting aid can be any organic solvent compatible with coating polymer solution, and it does not have negative impact to cross-linking reaction.Typical this solvent is the one of lower aliphatic alcohols, but also can use acetone, oxolane, acetonitrile and the easily miscible solvent of other and water.The amount of the organic solvent added is for by the minimum of coating solution at once required for wet porous matrix.The example of wetting aid comprises methyl alcohol, ethanol and isopropyl alcohol.
The method of brushing can improve the wetting of net by coating solution.Coating solution is made a forcible entry in net in a controlled fashion to soak into net uniformly and can not remain hydrophobicity spot or region.Pass through to apply the pressure extruded to make solution and enter net, this can by such as from gap pressure network or closely net and solution is extruded realization.Those skilled in the art can determine the condition of pressure, speed and the geometry prepared required for uniform coating.
The step preferably comprised for the formation of the method for the matrix of brushing is: 1) prepare solution; 2) solution is applied on film; Excessive liquid is removed from the outer surface of matrix; 3) desciccator diaphragm; 4) cured film; 5) also desciccator diaphragm is rinsed; 6) last film malleableize is optionally made; And 7) optional acid treatment film.More particularly, solution is prepared into containing suitable polymer and crosslinking agent.The concentration of these two components decides thickness and the swellbility of deposited coatings, and this defines by the flux of film and its adsorption capacity conversely, as follows.Polymer and crosslinking agent dissolve in a suitable solvent, are preferably water.Solution optionally can contain other composition, such as wetting aid, dispersing aid and pH adjusting agent.If use hydrophobic medium, the surface tension of solution must be low to enough soaking it.Aqueous solutions of polymers typically can not soak dewatering microporous film, therefore organic solvent (wetting aid) must be joined in solution.Be dispersed on the surface of hydrophobic membrane in order to what help coating uniform, surfactant can be added in solution.Finally, depend on the chemical characteristic of crosslinking agent, need to raise pH value to realize cross-linking reaction.Solution component and typical concentration range are listed in table 1:
table 1
| Component | Function | Scope, wt.% |
| Polymerism primary amine | The adsorpting polymerization thing that hydrogel is formed | 3-15 |
| Crosslinking agent | Affect the formation of hydrogel | 0.01-2.0 |
| Surfactant | For the surfactant making coating smooth | 0-3.0 |
| Hydrophilic polymer | Surface hydrophilic | 0-1.0％ |
| Organic solvent | With coating solution initial wetting hydrophobic film | 0-30 |
| Inorganic base | Raise pH value crosslinked to affect | 0-5.0 |
| Water | Solvent | Balance |
Coating solution is applied in matrix, such as, by by matrix submergence in the solution, from solution, removes matrix and mechanically remove unnecessary solution from the both sides of matrix, such as, using a pair mip rolls (extruding out).Then dry hole is full of the porous matrix of solution.Drying can at room temperature be undertaken by evaporating or can passing through application of heat (temperature range of about 40-110 DEG C).After matrix drying after brushing, kept a period of time of several hours to several days so that crosslinking agent can fully and polymer reaction.Being cross-linked can optionally through the hot acceleration of application.Use a large amount of solvent washing matrix afterwards and drying again.The film absorber that the step of additional optional comprises at elevated temperatures the drying of (60-120 DEG C) malleableize is to regulate its flowing property and it makes the protonated amino be present in coating with the concentration strong non-ionic monobasic acid process that is 0.1M to 1M.
When polymer is PAA, amino all in polymer being converted into substantially corresponding ammonium salt after heating film will contribute to ensureing the robustness of product.Good wettability is important.Because basic material is very hydrophobic and hydrophilic coating is very loose crosslinked and it is not be connected in matrix covalency, therefore the shrinkage of the PAA gel of some sides will cause that film is become and can not use water-wet.On the other hand, if all amino of PAA are all converted into corresponding ammonium salt substantially, volume, the larger water confining force produced by counter ion that dry coating increases and will to contribute to that film is more added water for the stronger affinity of the water of electropolymer wettable.For avoiding PAA ionomer, in order to this object should use strong, nontoxic, non-oxide acid, this acid being preferably monovalence makes PAA protonated.Suitable acid comprises hydrochloric acid, hydrobromic acid, methanesulfonic acid, sulfamic acid, trichloroacetic acid and trifluoroacetic acid.Although chloride can be the counter ion selected, because it is Already in sample protein matter solution, for continuous print process, use hydrochloric acid and/or its salt to be unpractical, this is because it is to the corrosivity of steel and the employment security problem included by it.Therefore more suitably acid is carbamic acid (H
2n-SO
2oH), it is preferably as the protonating agent of PAA.
The suitable method for protonated PAA is be immersed in the solution of 0.1-0.5M Bronsted acid by film, is preferably sulfamic acid in water (or water/alcohol mixture is so that fully infiltration is difficult to the film that soaks), afterflush and drying.The film obtained will with being easy to by using the experimental program of single condition to exchange the sulfamic acid counter ion of falling, and the NaOH of such as 0.5M is the sodium chloride of 0.5M afterwards.
This acid treatment improves the pot-life stability of film, and causes significantly higher bond strength.Although the present invention is not limited to any special theory, but it is believed that when PAA is when the state of completely protonated (acid-treated) is dried, it shows form that is wider, open to the outside world, it has the ability encapsulating BSA better, and can not be discharged before reaching higher ionic strength thus.The further benefit of acid treatment film has larger stability for ionizing irradiation, such as the known γ radiation for screening product sterilisation process.
Define successful film absorber product and have three crucial parameters.They are: adsorption capacity, flux and bond strength.And bond strength is determined by the characteristic of the group be present on film absorber surface to a great extent, capacity and flux usually to for the formation of the process of adsorption layer and the amount of polymer and crosslinking agent more responsive.Often can observe the chemical characteristic for given support frame compound, purification efficiency (determined by the height of bed) and film absorber, higher flux can be converted into less adsorption capacity, and vice versa.
Figure 1A and 1B shows the typical tendency of the film absorber prepared in accordance with the present invention observed.As can be seen from these figures, the balance between flux and capacity is obvious.Can find out that PAH and crosslinking agent these key property on film absorber have direct impact.In order to operation satisfied in the application, good film absorber should have high flux and high power capacity.The flux of film absorber can be expressed with chromatography unit CV/min/psi usually, and wherein CV is column volume.The flux of given column volume obviously depends on the post height that usual optimization is effectively separated.When anion-exchange membrane absorber, effective post is high can by realizing the electronegativity virus of removing (LRV4.0) 99.99%, such as, minimum constructive height definition required for MMV.Find that for this post height film absorber of the present invention be about 0.5mm.Disposing virus to more guarantee, running through the pillar that the present invention's routine uses double height size (1mm).Having flux that the high film absorber of this post expects should be at least 2.0CV/min/psi or more, is more preferably 2.5CV/min/psi or more.The combination obtaining suitable flux and capacity is addition difficult.The present inventor must exceed normal experiment significantly or pay close attention to optimization to obtain the character of outstanding film absorber.Such as, wisely crosslinking agent is chosen as highly flexible, polymerism molecule and proves that it is highly profitable to this adsorption capacity.The PAA (15000) of intermediate molecular weight is used to allow to obtain the balance between flowing and capacity.Inorganic base for adjust ph is non-class of saltouing, and therefore the PAA of higher concentration can be used for obtaining high power capacity.The selection of surfactant by confirm required nonionic, the compound of corrosion stable is controlled, and the amount of surfactant needed for finding is in an independent research.On the contrary, the PAA film absorber prepared according to the application US2005/0211615 do not authorized is for the flux only having 0.5CV/min/psi 1mm post, and BSA capacity is only about 61mg/ml.
Another important aspect of the present invention is the last handling process used after the solidification of absorber film, flushing and drying.Inventor finds to process the film absorber based on polymerism primary amine with acid to be a significant increase bond strength, wetability and the stability for ionizing irradiation.
One of inventor is very wonderful is found to be the bond strength that acid treatment is a significant increase film absorber, increases to 78-82mS/cm from about 54-58mS/cm.Can suppose that it shows the wider open to the outside world form better can encapsulating BSA when PAA is when the state of completely protonated (acid-treated) is dried, and thus until higher ionic strength time just can discharge it.These benefit right and wrong are calculated and wonderful, but are usually converted into the trace impurity more easily removed due to higher SB, and therefore this is highly profitable.
The permeability of crosslinked PAA film absorber is improved by high temperature " solidification " process.Lightly crosslinked PAA gel has the ability absorbing large quantity of moisture, and this causes the order of magnitude of its volume to increase.This result can cause low permeability.It seems, this character of gel reduces by making it be dewatered to a certain degree, and described degree makes swellingly be reduced to acceptable level and do not damaged bond strength and the capacity of gel.In fact, it is necessary for can adjusting permeability of the membrane to solidification process product.Suitable solidification temperature is about 25-120 DEG C, is more preferably about 85-100 DEG C, solidifies about 6 to 72 hours.
γ radiation is the sterilization process for screening product accepted extensively.γ sterilization capability is the feature of the film absorber expected.It is that acid-treated film absorber has the better stability of ionizing irradiation in practice that inventor observes wonderful benefit.Fig. 2 show film absorber sample (control, without acid treatment) and those be converted into the BSA capacity of Amcide Ammate sample.All these samples all use the electron beam irradiation of 25kGy to simulate γ sterilising conditions.Clearly acid-treated sample is than better not maintaining their character with acid-treated sample.
Therefore, when being prevented in Mab purification layer analysis system, the anion exchange absorbing device of formation is useful especially.Such as, the cell culture fluid comprising monoclonal antibody can use affinity stratographic analysis purification, such as a-protein or Protein G affinity chromatography, is one or more cation exchange columns afterwards.Then the output from last cation exchange column can flow through anion-exchange column of the present invention, by making these impurity be combined with medium under suitable condition for reducing in fluid the concentration remaining host cell proteins matter, virus, nucleic acid, endotoxin and other impurity significantly, thus product that is useful, purifying is allowed to flow through interchanger.
Importantly, use the cation exchange pond of quick anion exchange absorbing device can realize further purifying (output from cation exchange column) and there is no the dilution in significant cation exchange pond, in order to reduce the concentration (with the conductivity reduced) of salt to make host cell proteins matter effectively combine, this is normally necessary.In fact, absorber of the present invention even still has high in conjunction with flux (such as in 200mMNaCl, the BSA of >60g/L combines) under high salt concentration and conductivity, and under the condition of typical cation exchange pond, allow a lot of quality bearing capacity (3kg/L such as with 0.05kg/L compared with) higher than conventional absorber, see following examples 7.Due to quick adsorption device high power capacity under high salt concn, it reduces or eliminate the ability of diluting in cation exchange pond is significant advantage.
In relatively high salinity with under relative high pH value, absorber of the present invention also shows strong removal (still obtaining the removing of acceptable virus under the pH value of 7.6 and the salinity of 100mM and 150mM) to virus.
Those skilled in the art also recognizes, in some applications, the downstream polishing undertaken by one or more cation exchange columns may be unnecessary, and anion exchange absorbing device can be placed on the downstream of affinity post and there is not middle cation exchange column betwixt fast.Similar, for some application, quick anion exchange absorbing device can be placed on the downstream of cation exchange column and catch (affinity) post before not needing it.
Here the following embodiment comprised is not inclined to restriction the present invention for illustration of object of the present invention.
Embodiment 1 (PAA on hydrophobicity UPE)
Preparation is containing the isopropyl alcohol of 20wt.%, the PAH (molecular weight 15 of 9wt.%, 000), the PEG-DGE (molecular weight 526) of a hydronium(ion) lithia of 3wt.%, TritonX-100,0.5wt.% of 2wt.% and the polyvinyl alcohol (molecular weight 30 of 0.2wt.%, the degree of hydrolysis of 000,98%) the aqueous solution.Be this solution complete wetting of hydrophobicity UPE film of 0.65 μm by pore-size grade, and the extrusion of excessive liquid is fallen.Film at room temperature dry 24 hours is also again dry with a large amount of water and washed with methanol.Film is spontaneously wet out by water easily and increases by the weight of about 25%.Eight layers that manufacture with this film
type injection device has 3.5cm
2surface area and 1mm post high.This device has the bond strength of the flow rate of 4.3CV/min/psi, the BSA capacity of 90mg/ml and 54mS/cm.
Comparative example 1 (PAA-GTMAC on hydrophobicity UPE)
From the Modified Membrane of embodiment 1 by being immersed in the glycidyltrimetiiylammonium ammonium chloride (GTMAC) of 50wt.% 24 hours when pH value 13, rinse with water and dry and modification further.Eight layers that manufacture with this film
type injection device has 3.5cm
2surface area.This device has the bond strength of the flow rate of 0.7CV/min/psi, the BSA capacity of 80mg/ml and 19mS/cm.
Comparative example 2 (sulfamic acid process)
From embodiment 1 Modified Membrane by the aqueous sulfamic acid that is immersed in 0.5M 10 minutes, with deionized water rinsing and dry and modification further.Eight layers that manufacture with this film
type injection device has 3.5cm
2surface area.This device has the bond strength of the flow rate of 4.3CV/min/psi, the BSA capacity of 80mg/ml and 80mS/cm.
Comparative example 3
The aqueous solution of the Aqueous polyvinylamine solutions of the chloropropylene oxide modification of the sodium hydroxide solution of preparation containing the PAH (molecular weight 75,000) of 11.6wt.%, the 1.0N of 18.6wt.%, the sodium chloride of 11.6wt.%, 17% of 23.2wt.%.By pore-size grade be the hydrophobicity UPE film methyl alcohol pre-wet of 0.65 μm and directly contact 5 minutes with this solution.Wet film to be placed in the baking oven being placed on 85 DEG C in polyethylene film bag and by film bag 7 minutes, carefully not to make film parch to cause cross-linking reaction.Then wet film shifted out from bag and allow at room temperature dry.Afterwards the film of drying to be placed in the baking oven of 100 DEG C 4 hours to complete cross-linking reaction.Then the thoroughly washing film also permission at room temperature drying of water, methyl alcohol and hydrochloric acid (1.0N) is used.Film without water-wet.Eight layers that manufacture with this film
type injection device has 3.5cm
2surface area.This device has the bond strength of the flow rate of 0.5CV/min/psi, the BSA capacity of 61mg/ml and 81mS/cm.
Embodiment 2 (PAA on hydrophily UPE)
The aqueous solution of preparation containing the PAA (molecular weight 15,000) of 10wt.% and the PEG-DGE (molecular weight 526) of 0.5wt.%.Be this solution complete wetting of hydrophily UPE film of 0.65 μm by pore-size grade, and the extrusion of excessive liquid is fallen.Film at room temperature dry 24 hours, rinses with a large amount of water and drying again.Eight bed devices have the bond strength of the flow rate of 4.3CV/min/psi, the BSA capacity of 80mg/ml and 49mS/cm.
Embodiment 3 (polyvinylamine on hydrophily UPE)
The aqueous solution of the PEG-DGE (molecular weight 526) of preparation containing the isopropyl alcohol of 20wt.%, the polyvinylamine (Lupamin9095, BASFCorp., MountOlive, NJ) of 7wt.% and 0.5wt.%.Be this solution complete wetting of hydrophily UPE film of 0.65 μm by pore-size grade, and the extrusion of excessive liquid is fallen.Film at room temperature dry 24 hours is also again dry with a large amount of water and washed with methanol.Eight bed devices manufactured with this film have 3.5cm
2surface area.This device has the bond strength of the flow rate of 3.1CV/min/psi, the BSA capacity of 87mg/ml and 32mS/cm.
Embodiment 4 (polylysine on hydrophobicity UPE)
Preparation is containing the isopropyl alcohol of 20wt.%, the polylysine hydrobromide (molecular weight 30 of 4wt.%, 000-50,000), the aqueous solution of the PEG-DGE (molecular weight 526) of a hydronium(ion) lithia of 4wt.%, TritonX-100,0.25wt.% of 1wt.% and the polyvinyl alcohol (molecular weight 30000, the degree of hydrolysis of 98%) of 0.1wt.%.Be this solution complete wetting of hydrophobicity UPE film of 0.65 μm by pore-size grade, and the extrusion of excessive liquid is fallen.Film at room temperature dry 24 hours is also again dry with a large amount of water and washed with methanol.Film does not have with water-wet and has the weight increase of about 9%.Eight bed devices manufactured with this film have 3.5cm
2surface area.This device has the bond strength of the flow rate of 7.1CV/min/psi, the BSA capacity of 34mg/ml and 32mS/cm.
Embodiment 5 (PAA on activated agarose bead)
With PAA modified agarose chromatography beads.The Epoxy Sepharose 6B (GEHealthcare) of 2g is suspended in the Milli-Q water of 8ml, adds PAA (molecular weight 3, the 000) solution of 10ml10% wherein.With NaOH, pH value is adjusted to 11.By bead vibration gently 24 hours, the flushing careful with water and before use in refrigerator hygrometric state store.Pile up chromatographic column (post height 5.5cm, column volume 1.88mL) with the bead of modification and test.It has the BSA capacity of 20.6mg/ml and the BS of 83mS/cm.
Comparative example 4 (PAA-GTMAC on activated agarose bead)
The agarose bead of the PAA modification from embodiment 5 and the GTMAC solution of 2% are reacted for 11 times in pH value and spends the night and modification further.With the flushing bead also hygrometric state storage in refrigerator before use that water is careful.Pile up chromatographic column (post height 4.7cm, column volume 1.61mL) with the bead of modification and test.It has the BSA capacity of 87.3mg/ml and the BS of 25.4mS/cm.
Embodiment 6
The film with the PAA of grappling from embodiment 1 to be sealed in 8 layers of injection device and Test Virus retention rate.Show to increase salinity to the impact of MMV and MuLV retention rate with the data in following table 2.It is as shown in the table, the MMV data of PallMustang film (bond strength of 20mS/cm) show that LRV reduces along with the rising of conductivity, and even still can observe and remove completely under the salinity raised for film of the present invention (i.e. high bond strength).
In addition, the HCP removing ability of PAA film is better than quaternary ammonium salt chemistry (PAA-GTMAC manufactures according to comparative example 1), significantly see table 3.For the data in table 3, governing factor is the CaptoQ with moderate binding strength (i.e. about 30mS/cm) that GEAmersham sells.It should be noted that the acquisition of these LRV does not have obvious loss of product because monoclonal antibody is to the non-specific binding of functional membrane (namely effective opposing is from the anion-exchange property of film surface species).
table 2Aas the removal (X-MuLV) of the virus of salinity parameter
table 2Bas the removal (MMV) of the virus of salinity and pH parameter
table 3hCP is removed from three kinds of different monoclonal antibodies
| Product (Pro.APool) | Conductivity (mS/cm) | HCP bearing capacity (ng/ml) | The removal (@400CV of prototype (PAA from embodiment 1)) | The removal (@30CV of CaptoQ) | The removal (@400CV of quaternary ammonium salt) |
| 4g/LMab1 | 10.7 | 420 | 62％ | 14％ | 0％ |
| 7g/LMab2 | 9.7 | 1800 | 31％ | 0％ | 0％ |
| 3.7g/LMab3 | 22 | 200 | 85％ | 30％ | 0％ |
Embodiment 7
Controlling UPE film brushing machine being brushed 0.65 μm with the PAA of 9% and the PEG-DGE of 0.5%.Film is extracted, and at 85 DEG C in convection oven dry 10 minutes.Placed 12 hours at 85 DEG C by a sample, another sample at room temperature stores.Then in the isopropyl alcohol of 5%, film is processed with stable coatings with the sulfamic acid of 5%.Then permeability of the membrane is measured with flux tester.In this test, the time that the water that (27.0 " Hg) measure 500ml is under vacuo spent by membrane sample (cross section area 9.6sq.cm).Manufacture the film group listed by table 1, to prove the infiltrative improvement as curing schedule result.As seen from Table 4, " uncured " film ratio " solidification " film has noticeable higher flowing time (showing to reduce permeability).
As can be seen from Table 5, curing schedule can be used for desirably controlling permeability of the membrane and capacity.All films all have the BSA bond strength between 60-75mS.
Table 4: contrasting with the uncured membrane flow time of solidification
Type number condition of cure flux (s/500ml)
020307-60821-1180
020307-6082185 DEG C 12 hours 96
020307-60803-2650
020307-6080385 DEG C 12 hours 106
Table 5: for the treatment conditions of film absorber
| UPE type number | PAA type number | Roller is numbered | Solidification temperature (DEG C) | Hardening time (hour) | Flowing time (s/500ml) |
| UPDP101006R9 | 60821 | 103A | 100 | 16 | 58 |
| UPDP101006R9 | 60821 | 103B | 90 | 6 | 95 |
| UPDP101006R6 | 60803 | 104 | 90 | 6 | 102 |
| UPDP101106R5 | 60821 | 105 | 90 | 6 | 90 |
| UPDP101106R5 | 60801 | 102 | 90 | 6 | 84 |
| UPDP101006 | 60821 | 011507-1 | 90 | 12 | 80 |
| UPDP101106R5 | 60821 | 011507-2 | 95 | 12 | 52 |
Embodiment 8
Controlling UPE film roller brushing machine being brushed 0.65 μm with the PAA of 9% and the PEG-DGE of 0.35%.With water and methyl alcohol, film is extracted, and dry at 120 DEG C in hot air impingement drier.Then roller is made to solidify under different temperatures as shown in the table and time.Then in the isopropyl alcohol of 5%, film is processed with stable coatings with the sulfamic acid of 5%.Then permeability of the membrane is measured by the method that flux tester describes according to embodiment above.
As shown in the table, the flowing time in 50s rank can by heating 12 hour or longer time at the temperature of 90 DEG C by film and obtain being much higher than.When expect there is the film of comparatively hypotonicity (the higher flowing time in 100s rank), can by temperature limiting to shortening the heat time (<10h) lower than 85 DEG C.
| Capacity, flowing time (mg/ml, s) | 8 hours | 10 hours | 12 hours | 20 hours | 24 hours |
| 85℃ | 107.7，147.6 | 95.63，9 | 92.9，88.0 | ||
| 90℃ | 95.41 | 90.39 | |||
| 95℃ | 92.6，79.2 | 89.6，50.2 | 85.8，67.1 | ||
| 100℃ | 79.39 | 63.41 |
Claims (26)
1. a porous adsorbing medium, it comprises porous matrix, described porous matrix has two surfaces relevant to the geometry of matrix and the dimension of thick layer between two surfaces is porous, wherein said geometric jacquard patterning unit surface refers to outer surface, the surface area relevant to hole refers to interior surface area, wherein with loose crosslinked adsorptivity hydrogel coating internal surfaces and outer surface, so that described hole can not be blocked completely, wherein coated polymeric defines adsorptivity hydrogel, and crosslinking agent and described coated polymeric react and make described coated polymeric water insoluble, wherein said coated polymeric is selected from polyallylamine, polyvinylamine, poly-butylamine, polylysine, they each other and with the copolymer of other polymer, and their corresponding protonated form,
Wherein crosslinked described coated polymeric is effectively can produce the flowing of at least 2.0CV/min/psi, the amount existence of the BSA of about 60g/L combines in the NaCl of 200mM binding capacity by the 1mm floor height of described medium.
2. the porous adsorbing medium of claim 1, wherein crosslinked described coated polymeric is effectively can produce the amount existence of the flowing of at least 2.5CV/min/psi by described medium.
3. the porous medium of claim 1, wherein said matrix contains microporous barrier.
4. the porous medium of claim 3, wherein said film contains polyolefin.
5. the porous medium of claim 4, wherein said polyolefin is polyethylene.
6. the porous medium of claim 1, wherein said matrix is ultra high molecular weight polyethylene films.
7. the porous medium of claim 1, wherein said polymer contains polyallylamine or protonated polyallylamine.
8. the porous medium of claim 1, wherein said polymer comprises copolymer containing polyallylamine or protonated polyallylamine or block copolymer.
9. the porous medium of claim 1, it has the nucleic acid binding ability higher than 5mg/ml.
10. the porous medium of claim 9, wherein said nucleic acid is DNA.
11. an anion exchanger, it comprises the outer cover containing, for example porous adsorbing medium according to claim 1.
12. the anion exchanger of claim 11, wherein said matrix is UPE film.
The anion exchanger of 13. claims 11, wherein said polymer contains polyallylamine or protonated polyallylamine.
14. 1 kinds of systems for purifying biological solution, it comprises affinity chromatography separator, the anion exchanger at least one cation exchanger and described cation exchanger downstream, described anion exchanger is containing, for example porous adsorbing medium according to claim 1.
15. the system of claim 14, wherein said medium is positively charged.
16. the system of claim 14, wherein said affinity chromatography separator contains protein A affinity post.
17. 1 kinds of purifying contain the method for the biological sample of antibody and impurity, and described method comprises: stand affinity chromatography by making described sample and biological sample described in purifying; Described sample is further purified afterwards by the anionic exchange medium that makes described sample be carried on containing, for example porous adsorbing medium according to claim 1; And collect the sample be further purified.
18. 1 kinds of purifying contain the method for the biological sample of antibody and impurity, and described method comprises: stand cation-exchange chromatography by making described sample and biological sample described in purifying; Described sample is further purified afterwards by the anionic exchange medium that makes described sample be carried on containing, for example porous adsorbing medium according to claim 1; And collect the sample be further purified.
The method of 19. claims 17, is carried on described anionic exchange medium after being included in described affinity chromatography steps further and by described sample and carries out cation exchange.
The method of 20. claims 17, wherein said biological sample is cell culture medium.
The method of 21. claims 17, the wherein said sample collection be further purified is in the wasteway of described anion exchanger.
22. 1 kinds of methods manufacturing absorber film, comprise and a kind of porous matrix is provided, described porous matrix has two surfaces relevant to the geometry of matrix and the dimension of thick layer between two surfaces is porous, wherein said geometric jacquard patterning unit surface refers to outer surface, the surface area relevant to hole refers to interior surface area, wherein with loose crosslinked adsorptivity hydrogel coating internal surfaces and outer surface, so that described hole can not be blocked completely, wherein coated polymeric defines adsorptivity hydrogel, and crosslinking agent and described coated polymeric react and make described coated polymeric water insoluble, and the matrix obtained is heated to the temperature of 25 DEG C to 120 DEG C, wherein said coated polymeric be selected from polyallylamine, polyvinylamine, poly-butylamine, polylysine, they each other and with the copolymer of other polymer, and their corresponding protonated form, wherein crosslinked described coated polymeric is effectively can produce the flowing of at least 2.0CV/min/psi, the amount existence of the BSA of about 60g/L combines in the NaCl of 200mM binding capacity by the 1mm floor height of described medium.
The method of 23. claims 22, wherein said temperature is 85 DEG C to 100 DEG C.
The method of 24. claims 22, comprises the film obtained with acid treatment further, and makes acid-treated film stand gamma-rays.
The method of 25. claims 24, wherein said acid is selected from the group be made up of hydrochloric acid, hydrobromic acid, sulfamic acid, methanesulfonic acid, trichloroacetic acid and trifluoroacetic acid.
The method of 26. claims 24, wherein said acid is sulfamic acid.
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| EP2570182A1 (en) * | 2011-09-15 | 2013-03-20 | InstrAction GmbH | Sorbent comprising on its surface a cationic or protonizable aliphatic residue for the purification of organic molecules |
| FR2991689B1 (en) * | 2012-06-11 | 2018-04-20 | Diagast | IMMUNO-HEMATOLOGICAL DIAGNOSTIC DEVICE AND USES |
| CN102786577B (en) * | 2012-07-20 | 2014-07-09 | 中国医学科学院输血研究所 | Simple device for preparing plasma protein products through batch adsorption, and method thereof |
| CN109078505A (en) * | 2018-08-24 | 2018-12-25 | 杭州九龄科技有限公司 | A kind of composite chromatography filter membrane and its preparation method and application containing adsorbing medium |
| CN109806916B (en) * | 2019-03-15 | 2021-12-21 | 中科森辉微球技术（苏州）有限公司 | High-performance anion exchange medium and preparation method thereof |
| CN117169393B (en) * | 2023-11-03 | 2024-03-19 | 杭州湃肽生化科技有限公司 | Method for detecting cyclic peptide in plant tissue |
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| SE9600590D0 (en) | 1996-02-19 | 1996-02-19 | Pharmacia Biotech Ab | Methods for chromatographic separation of peptides and nucleic acid and new high-affinity ion exchange matrix |
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