CN101027080A - Methods of protein fractionation using high performance tangential flow filtration - Google Patents

Abstract

Processes and apparati are provided for separating molecules of interest from a mixture containing them which comprises subjecting the mixture to an improved method of high performance tangential flow filtration (HPTFF). The HPTFF of the invention was used to clarify, and process various feedstreams for the removal of a molecule of interest based on both the size and charge of the molecule of interest. According to a preferred embodiment, a transgenic milk feedstream is stabilized and particulate matter such as fat, casein miscelles and bacteria are removed. The method of HPTFF used in the current invention utilizes optimized process parameters that include temperature, trans-membrane pressure, molecular charge, molecular size, cross-flow velocity, and milk concentration. Cleaning and storage procedures were also developed to ensure long membrane life. An aseptic filtration step was also developed to remove any bacteria remaining in a clarified transgenic milk feedstream.

Description

Carry out the method for protein fractionation with high performance tangential flow filtration

Priority

The application requires the priority of the USSN 60/550,137 of submission on March 4th, 2004, and its content is incorporated herein by reference.

Technical field

The invention provides a kind of improving one's methods and system of special target molecule of from the pollutant of initial charge is flowed, finding, clarifying.More specifically, method of the present invention provides the improve one's methods processing of sample solution by high performance tangential flow filtration, and wherein tangential flow filtration can strengthen clarification, the concentrated and classification of expectation molecule from given incoming flow.

Background technology

The present invention relates to a kind of from given incoming flow the improving one's methods of classification molecules of interest.Should be noted in the discussion above that the purified relatively in a large number bioactive molecule of production, is very important for pharmaceutical preparation, protein, enzyme, antibody and other specialty chemicals of making humans and animals economically.In many polypeptide, antibody and proteinic production, various recombinant DNA technologies have become the method for selection, because these methods can these protein of large-scale production.Various " platforms " that can be used in these productions comprise antibacterial, yeast, insecticide or mammalian cell cultivation and transgenic animal.For the transgenic animal system, the preferred animal type is to produce in mammal, but this platform production method also can consider to use birds or even transgenic plant produce exogenous protein, antibody or its fragment or fusions.

Produce recombinant protein and comprise, and host cell, transgenic animal or plant are grown under the condition that helps express recombinant protein matter or other molecules of interest with this protein DNA transfection host cell of coding.Prokaryote-escherichia coli are a kind of favourable host systems, because it can produce recombinant protein with higher yields.But, express for the generality of the DNA of coded protein and to have many United States Patent (USP)s, developed the multiple expression platform from escherichia coli to the cattle.

Improvement along with produce exogenous protein or other molecules of interest from biosystem has had the increasing pressure requirement development new technologies on industry, strengthen the purification of the biological product of production like this and medicine and reclaim technology and make it more efficient.That is to say that along with the increase of new product lines, people have very big interest design new method that these therapeutants are come into the market rapidly with commercial quantities.Simultaneously, also be faced with new challenges, need exploitation to comprise the new technology that reclaims transgenic protein and antibody milk, blood and the urine from various body fluid industrial.Production scale is big more, and these problems just often become complicated more.In addition, go back the challenge of demand side to product purity and safety, particularly virus safe and as residual contaminants such as DNA and host cells, it may need to satisfy the requirement of the various government organs of the biological serviceability drug manufacture of supervision.

Current, there has been several method to come from its mixture effectively separating bio to learn molecule (s) of interest, for example protein.An important techniques is an affinity chromatography, and it comes isolated molecule according to the specificity and the selective binding of expectation molecule and affinity substrate or gel, and the molecule of not expecting simultaneously keeps unbound state, can remove from system then.Affinity gel typically is made up of the ligand binding moiety that is fixed on the gel carrier.For example GB 2,178, and 742 utilize affinity chromatography to come purified hemoglobin and chemical modification derivant thereof, and it is based on the following fact: natural hemoglobin combines with the polyanion structure specificity of specific family.These structures are fixed in gel and originally are used on one's body catch.In the method, the hemoglobin of unmodified remains on the affinity gel, and therefore the hemoglobin of modifying can not combine with gel and remove from system because the polyanion binding site is occupied by the dressing agent covalency.Affinity chromatographic column is a high degree of specificity, so just can obtain very pure product; But affinity chromatography is relatively costly method, and it is very difficult therefore using it for commercial operation.

Typically, purification genetic engineering bio-pharmaceutical from the supernatant that comprises various different host cell pollutant.Reversed-phase high-performance liquid chromatography (RP-HPLC) can be used for protein purification, because it can high efficiency separation structure or the similar especially molecule of molecular weight.Disclose the method for utilizing RP-HPLC and can be used for many molecules.McDonaldand Bidlingmeyer，″Strategies for Successful Preparative Liquid Chromatography″，PREPARATIVE LIQUID CHROMATOGRAPHY，Brian A.Bidlingmeyer(New York：EIsevier Science Publishing，1987)，vol.38，pp.1-104；Lee et al.，Preparative HPLC.8th Biotechnology Symposium，Pt.1，593-610(1988)。

In addition, in the face of in another industry of some identical challenges, also need new answer.Since begin to be used for to concentrate and the classification milk surum and handle waste water after, dairy industry is exactly to adopt this technology to use film system to come one of supporter of classification, clarification and purification.In the eighties in 20th century, the researcheres of dairy industry begin to use in the nonstandardized technique cheese production film to concentrate breast.In recent years, technological improvement makes the membrance concentration breast compare more attractive in the past.Simultaneously, the technological progress of membrane material, process engineering and newborn component function makes that membrane separation process is all more practical and more useful in almost each stage that breast is handled.Though these practices can not be used for all aspects of dairy industry, their potentiality are huge.

For example, be attractive especially at following membrance separation for fluid breast processor, because it needs energy hardly, during handling, can not destroy any product.Every kind is used for different purposes to 4 kinds of potential dairy industrys that are used for of underlying membrane filtration types---reverse osmosis (PC), nanofiltration (NF), ultrafiltration (UF) and microfiltration (MF)---.Some are used to handle and only relate to single film of planting; But advanced technologies is just using two or more films to handle in given application.Yet,, remain limited for some aspects of the bio-pharmaceutical production of dairy industry, food manufacturing industry and transgenic animal though these processing are useful.

In biotechnological industries and dairy industry, traditionally ultrafiltration is used for based on the size separation protein mixture, wherein the ratio of protein molecular weight must be at least about 10 to 1.In a lot of commercial Application of industrial circle, particularly reclaim in the bio-pharmaceutical in the transgenic animal Ruzhong, and this is a restrictive factor.A lot of researchs betide during ultrafiltration system optimizes, and reach higher selectivity people such as (, (1997)) Van Reis by changing physical and chemical condition (being pH and ionic strength).The method according to this invention has been made more improvement to condition optimizing on pH and ionic strength direction, make to develop high performance tangential flow filtration (HPTFF) in the multiple incoming flow that comprises milk.

HPTFF utilizes multiple phenomenon to make the grading effect maximization.It comprises that working solution pH and ionic strength make the difference maximization of solute effective volume, and uses the film with controlled pore size.

As described, current industry and bio-pharmaceutical technology often use purification, concentrate and three separating steps of buffer-exchanged in often use ion exchange chromatography, UF and size exclusion chromatograph (SEC).Even but to interosculate, the separating power of these technologies also is limited.Even ultrafiltration (UF) is limited to the solute that separates at least 10 times of difference in size usually.In addition, the similar also extremely difficult separation of molecule of electric charge.HPTFF is two-dimentional purification process, and it utilizes the size of biomolecule and the difference of charge property.Therefore it might separate the biomolecule with same molecular amount.It in addition might keep a kind of biomolecule, allowing more simultaneously, the molecule of macromolecule passes through film.

Difference in size can be separated with hydrodynamics by using high selectivity charged membrane and the careful buffer of optimizing less than triple molecule.In HPTFF, the knowledge of the isoelectric point, IP of the molecules of interest of expectation (pI) is principal element.This will determine intrinsic charge characteristic curve, aperture and the flow performance of film formation and film then.In addition, HPTFF makes and might carry out all these steps in the individual unit operation, therefore reduced production cost.In addition, HPTFF uses the identical linear amplification principle of having set up in UF.Also can assist HPTFF by optimizing transmembrane pressure.

According to the type of film, can be divided into microfiltration or ultrafiltration.Micro-filtration membrane, aperture typically are used for clarification, degerming, removing microgranule or harvesting between 0.1-10 μ m.Ultrafilter membrane has much smaller aperture between 0.001 to the 0.1 μ m, is used to isolate and concentrates dissolved molecule (protein, peptide, nucleic acid, carbohydrate and other biological molecule), is used for exchange buffering liquid and is used for rough classification.

But there is restriction in the attainable protein purification degree of ultrafiltration.These restrictions mainly are because the pore-size distribution broad of concentration polarization phenomenon, obstruction and most of films.Therefore, the solute separating capacity is often lower.Referring to, Porter for example, ed., HANDBOOK OF INDUSTRIAL MEMBRANE TECHNOLOGY (NoyesPublications, Park Ridge, N.J., 1990), pp.164-173.The polarization layer effect of solute is equivalent to the additional filtering device, and connects with the primary ultrafiltration device in fact.This effect has produced filtering the remarkable resistance of given solvent.Degree of polarization can cause a lot of surface abnormalities or uncertain effect along with the concentration of the solute that keeps increases and increases in real system in charging.For example, under the high degree of polarization condition, filtering speed only increases slightly along with the increase of pressure, comparatively speaking, under the non-polarized condition filtering rate usually and pressure linear.Use more open, more high-throughout film may not increase filtration rate, because polarization layer provides filtering restriction resistance.Interaction between reservation and the eluted solute makes this situation further complicated.The result of the concentration polarization and the process of obstruction makes and can not effectively utilize the macromole classifying capability of ultrafilter membrane to be used for extensive classification macromole mixture, for example plasma protein.Referring to Michaels, " Fifteen Years of Ultrafiltration:Problems and Future Promises of anAdolescent Technology ", in ULTRAFILTRATION MEMBRANES ANDAPPLICATIONS, POLYMER SCIENCE AND TECHNOLOGY, 13 (Plenum Press, N.Y., 1979, Anthony R.Cooper, ed.,), pp.1-19.

TFF and HPTFF can be according to further segmentations of the size of separating the level component.Handle the size that its scope can be from the intact cell to the buffer salt for protein.Below table 1 typical component that described that tunicle keeps in every kind of group in detail and by film.In addition, it has shown the scope of the membrane aperture classification that falls into each group usually or nominal molecular weight restriction (NMWL).

	Microfiltration	Virus filtration	High efficiency filter	Ultrafiltration TFF	Nanofiltration/reverse osmosis
						The film retained fraction	The intact cell cell debris	Virus	Protein	Protein	Antibiotic sugar salt
Film film film film film film film film film film film film film film film film film film film film film film film film film film film film film film
						See through the component of film	Colloidal substance virus protein salt	Protein salt	Protein salt	Little peptide salt	(salt) water
The scope of roughly damming of film	0.05μm- 1μm	100kD- 0.05μm	10kD-300kD	1kD- 1000kD	＜1kD

Table 1: the segmentation of tangential flow filtration method

It is known using tangential flow filtration to come separate substance.People's such as Marinaccio U.S. Pat 4,888,115 has disclosed the method (being called " cross-flow ") that is used for separating bio liquid and for example is used for the blood constitutent of plasmapheresis.In the method, blood is tangentially by (promptly striding across) organic polymer microporous filter membrane, and removes particulate matter.In another example of this area, disclosed tangential flow filtration has been used for filtered beer solution (Shackleton, EP 0,208,450, is disclosed on January 14th, 1987), especially for removing particulate matter such as yeast cells and other suspended solids.People such as Kothe (United States Patent (USP) 4,644,056, be issued on February 17th, 1987) disclosed this method of use from breast or first Ruzhong purification immunoglobulin, Castino (United States Patent (USP) 4,420,398, be issued to nineteen eighty-three December 13 days) described it and be used for separating for example interferon of antiviral substance from the meat soup that contains their cell culture residue of antiviral substance and virion and generation thereof.

The TFF unit has been used for from cell debris separation of bacterial enzyme (Quirk et al., 1984, ENZYMEMICROB.TECHNOL., 6 (5): 201).Use this technology, people such as Quirk can be with than extracting enzyme with conventional centrifugation technique higher yield and less time.Several application of tangential flow filtration have been done by Genovesi and have been summarized (1983 in pharmaceutical field, J.PARENTER.Aci.TECHNOL., 37 (3): 81), comprise and filter sterile water for injection, settled solution system and from meat soup and bacterial cultures, filter enzyme.

But required accurate control granular size is difficult in the commerce of this technology is used, usually also can be not successful.In the present invention, the use of tangential flow filtration is modified, and is used for coming separating particles in the effective and important technology of commerce according to size and electric charge.The HPTFF system of gained is used for the present invention, improves clarification and grading effect from the level that TFF realizes.Also disclosed and used selected big or small filter, further different big or small filters (being filtration system) are used in use or series connection successively, are used for the granule of separating particles with acquisition certain desired magnitude range.

Can be from cell culture or transgenic milk feedstream a kind of such molecules of interest of purification be people's alpha-fetoprotein of recombinating.Other molecules of interest include but not limited to, the Fc fragment and the fusion molecule of human albumin, antibody, antibody, wherein in fusion molecule human albumin or alpha-fetoprotein protein fragments as carrier molecule.

Method of the present invention also provides the precise combination of filter and condition, allows to optimize the productive rate of molecules of interest from given incoming flow.In these methods, important technical parameters such as pH and temperature are accurately operated.

Biological product industry is more and more paid close attention to Product Safety, purity and product cost.According to the present invention, using HPTFF is a kind of quick and more effective bio-molecular separation method.It can be widely used in biological field, for example immunology, protein chemistry, molecular biology, biochemistry and microbiology.

Description of drawings

Accompanying drawing 1 shows from incoming flow by the process chart of HPTFF to the Flow of Goods and Materials of loading and finishing.

Accompanying drawing 2A shows that the technology of microfiltration and equipment constitute.

Accompanying drawing 2B shows that the technology of TFF and equipment constitute.

The fluid flowing path that accompanying drawing 3 shows by different TFF and HPTFF module.

Accompanying drawing 4 shows the filtering technique flow chart.

Accompanying drawing 5 shows from DNA construct to producing the recombinate transgenic product development process of clarification breast of proteins of interest matter of Korea Spro.

Accompanying drawing 6 shows the equipment sketch map that is used for the inventive method.

Accompanying drawing 7 is presented in the HPTFF module type open and promotes turbulent feeding-passage.

Accompanying drawing 8 demonstrations HPTFF of the present invention system.

Summary of the invention

Sketch it, the objective of the invention is to use the HPTFF technology to realize more effectively protein fractionation.That is to say, use HPTFF to improve and from contaminating protein, separate proteins of interest matter.An example of proteins of interest matter is a recombinant human alpha-fetoprotein, and it is the protein of the about 66KD of molecular weight, has the structure similar to albumin.The target of method of the present invention is may keeping target protein (rhAFP) by effective method, and allows main infected milk protein pass through.According to the present invention, the lactoprotein of pollution comprises IgG, lactoferrin, albumin, casein, lactoglobulin and lactalbumin.According to an embodiment preferred of the present invention, effectively reduce whole concentration except that recombinant human alpha-fetoprotein and goat albumin with 100KD slipstream film method.By reducing the concentration of contaminating protein, the recombinant human alpha-fetoprotein of reservation purity and stable aspect all be enhanced, and can be further with conventional chromatogram method purification more effectively.

Method of the present invention is used rhAFP as an example, but also can be used for other protein of interest matter.When using the 20mM phosphate buffer to the solution continuous diffusion, use 100KD MWCO film to keep recombinant human alpha-fetoprotein, other protein can freely pass through this film then.

With SDS PAGE purity assay, be about 5-7% in the initial purity of clarifying the Ruzhong recombinant human alpha-fetoprotein.After the matter classification, relatively purity is brought up to approximately 30% at egg, and yield is 85%.This preliminary classification of the present invention has improved the efficient of downstream process because protein reach semipurified state quickened from multiple incoming flow preferably from transgene mammal milk to the processing of human therapy protein, protein fragments or antibody.

Therefore, in a preferred embodiment of the invention, the filtering technique that this paper developed and provided provide a kind of from breast natural constituents or its pollutant clarification, concentrate and the recombinant protein of classification expectation or the method for other molecules of interest.Clarifying a large amount of intermediate of gained are to be suitable for traditional purification technique such as stratographic feed material, and wherein chromatography is handled the downstream use at HPTFF, makes product reach final preparation and purity.

A preferred version of the present invention utilizes three filter element operations clarification from the given transgenic breast volume that contains molecules of interest, concentrates and this product of classification.Clarification steps is removed bigger particulate matter from product, for example Oil globule and casein micelle.Most micromolecule is removed in concentrating with classification step thereafter, comprises lactose, mineral and water, improves purity and the volume that reduces the products therefrom compositions.The product of HPTFF technology is concentrated into proper level, is beneficial to best downstream purification and whole product stability.Then this enriched product aseptic filtration, to guarantee that biological load is minimum and to strengthen the long-time stability of product.A large amount of degree of purity of production will reach 65% to 85%, can comprise for example albumin, lactalbumin (beta lactoglobulin, alpha-lactalbumin and BSA) and low-level residual fat and casein.This partially purified product is the desirable initiation material that is used for conventional downstream chromatographic technique.

The mainstream product that can handle with the present invention is the proteins of interest matter that transgenic is produced, and include but not limited to: alpha-fetoprotein, IgG1 antibody, fusion rotein are (for example: erythropoietin-human albumin fusions-" HEAP " or human albumin-erythropoietin; Beta-interferon-alpha-fetoprotein fusions), Antithrombin III, α-1-antitrypsin, IgG4, IgM, IgA, Fc part, comprise the peptide that is bonded to immunoglobulin fragment or the fusion molecule of polypeptide.Can comprise recombinant protein, exogenous hormone, endogenous protein or inanimate object reactive protein that can treated afterwards recovery biological function with other protein that the present invention handles.In these are handled, include but not limited to human growth hormone, recombined human albumin, decorin (decorin), human a-fetoprotein urokinase, tPA and prolactin antagonist.

In addition, according to the present invention, salt (NaCl) concentration change and two diafiltrations (diafiltration) step and are used for improving the purity that can reach according to the inventive method unlike the prior art.

An object of the present invention is to provide more effective high performance tangential flow filtration method, be used for coming materials such as separating particles and molecule according to size, this method has selectivity to substances of interest, obtains the purification of the higher multiple of this material.

Another purpose provides improved filter method, comprises ultrafiltration, is used for biomacromolecules such as isolated protein, and this method makes the concentration polarization minimum and do not increase flux.

Another purpose provides filter method, and it can be according to the material of size separation difference in size less than 10 times, and need not dilute this mixture before filtration.

The purpose of these and other is conspicuous for those skilled in the art.By following detailed description to the preferred embodiments of the invention, and with reference to the accompanying drawings, other features and advantages of the present invention will become apparent.

DESCRIPTION OF THE PREFERRED

In this manual, following abbreviation has specified implication:

Abbreviation:

The BSA bovine serum albumin

The CHO Chinese hamster ovary cell

The CV cross-flow velocity

The DFF in-line filtration

DV diafiltration volume

The IEF isoelectrofocusing

GMH mass flux (gram/m ²/ hour)-also can make J _M

LMH liquid flux (liter/m ²/ hour)-also can make J _L

The LPM liter/minute

The M molar concentration

The MF microfiltration

NMWCO name molecular cut off

The standardized water penetration of NWP

PES gathers (ether)-sulfone

PH is used to describe the term of the hydrogen ion activity of chemicals or chemical compound according to known section mathematic(al) parameter

PPM 1,000,000/portion

SDS-PAGE SDS (sodium lauryl sulphate) polyacrylamide gel electrophoresis

The SEC size exclusion chromatograph

The TFF tangential flow filtration

The PEG Polyethylene Glycol

The TMP transmembrane pressure

The UF ultrafiltration

Terminological interpretation:

Clarification

From solution, remove particulate matter, so that solution can pass through 0.2 μ m film.

Colloid

Relate to and easily to pass the macromole of capillary wall.These chemical compounds produce swelling (being that they attract liquid) load, are applied to usually and recover intravascular volume and improve perfused tissue.

Concentrate

, increase with respect to micromolecular ratio except that anhydrating and micromolecule with film so that keep molecule.

Concentration polarization

Cause keeping molecule in film surface accumulation (gel layer) by the combination of multiple factor: transmembrane pressure, cross-flow velocity, sample viscosity and solute concentration.

Cross-flow velocity

Fluid is striden the speed at top, film surface.CF=Pi-Po, wherein Pi is a porch pressure, and Po is an outlet pressure, and it is relevant with the retention flow velocity.

Diafiltration (Diafiltration)

Washing by film, is stayed grading technology in the retention with interested than macromole than micromolecule.It is a kind of convenience and otherwise effective technique, be used to remove or exchange salt, remove detergent, separated free and not binding molecule, remove low molecular weight substance or change ion fast or the pH environment.This technology is typically used micro-filtration membrane, and micro-filtration membrane is used for removing product interested from slurry (slurry), and it is constant to keep slurry concentration simultaneously.

Incoming flow

Raw material or material solution for being used for technology or method comprise proteins of interest matter, also can comprise all contaminations, comprise microorganism, virus and cell fragment.The preferred incoming flow of the present invention is the transgenic breast that comprises interested exogenous proteins.

Leach logistics capacity (J)

The part of sample is the ratio by film.

Flow velocity (V)

Fluid is considered to flow rate of fluid by the speed on film surface.To measure the product flow with change in flow.Relation between these two variablees allows us to determine mobile optimal performance window.

Classification

According to physics or chemical constitution isolated molecule in advance.

Coagulate the chamber layer

The micro-thin layer of the molecule that can form at the film top.It can influence that molecule keeps and therefore reduce and leach logistics and move by blocking the film surface.

High performance tangential flow filtration

HPTFF be a kind of can according to the size and electric charge carry out the high-resolution technology of protein-Separation of Proteins, thereby obtain product yield similar and purification of factor to chromatograph.The film NMWL that is used for HPTFF is in 10kD to 300kD scope.

Fenestra order of magnitude (MPSR)

The fenestra order of magnitude typically provides with the micron value, represents that granule than this bigger grade keeps tunicle.

Name molecular cut off (NMWCO)

The size of specified ultrafilter membrane (kilodalton).NMWCO is defined as 90% molecular weight that is retained in the globular protein matter on the film.

The name molecular weight limit (NMWL)

Film hierarchy system, indication molecule amount be higher than NMWL major part dissolving macromole, and molecular weight be lower than NMWL some will be kept by the discussion film.

The mark difficultyization lures aqueous (NWP)

The water of determining with specific recirculation rate during TFF device initial clean leaches flow velocity.This value is used to calculate the recovery of film.

Molecules of interest

The molecule of preparation isolating granule or other kinds from the solution of fluid such as liquid form or suspension.Interested granule or molecule separate from fluid, in most cases separate from fluidic other granules or molecule.The big young pathbreaker of molecules of interest to be separated determines the aperture of stand-by film.Preferably, molecule (s) of interest is biological and biochemical source or produces by transgenic or in vitro method, and comprises protein, peptide, polypeptide, antibody or antibody fragment.The example in preferred feedstock stream source comprises that mammal milk, mammalian cell are cultivated and microbial cell is cultivated for example antibacterial, fungus and yeast.Should be noted in the discussion above that and treat that filtering solids comprises undesirable polypeptide, protein, cellular component, DNA, colloid, mycoplasma, endotoxin, virus, carbohydrate and other interested biomolecule, no matter and whether it is by glycosylation.

Tangential flow filtration

A kind of technology, the fluid mixture and the membrane plane that wherein comprise separation component to be filtered are tangent with circulation at a high speed, to increase the mass transfer coefficient of diffusion dorsad.In this filtration, it is poor to exert pressure along the length direction of film, flows through filter to impel fluid and can filter solute.This filtration is fit to carry out with batch process and continuous flow process.For example, can simultaneously the fluid by filter be discharged in the isolating unit continuously with solution repeatedly by film; Perhaps with solution by film once, the fluid by filter carries out downstream continuously.

Transmembrane pressure

Along filter membrane length direction applied pressure difference gradient, flow through filter to impel fluid and can filter solute.In the slipstream system, TMP is the highest in import department's (beginning of flow channel), and (end of flow channel) is minimum in the exit.Average pressure with import, outlet, filter object out hole calculates TMP.

Reclaim

Handle the amount of the recuperable molecules of interest in back.Usually represent with the percentage ratio or the yield of initial substance.

Retention

Sample can not may also be referred to as concentrate by the part of film.Retention is recycled during TFF.

The tangential flow filtration basis

Two important variablees are arranged: transmembrane pressure (TMP) and cross-flow flow velocity (CF) in all slipstream devices.Transmembrane pressure (TMP) is the power of actual promotion molecule by filter hole.The cross-flow flow velocity is the flow velocity that solution strides across film.It provide strength to clear away to block film and therefore reduce process efficiency than macromole.In fact, from sample charging reservoir, pump the film surface (cross-flow) that fluid feed strides across filter, and return sample charging reservoir as retention.Put on the back-pressure that keeps property management by clip and set up transmembrane pressure, order about the molecule littler and pass filter and enter and leach thing (or exudate) part than fenestra.This cross-flow cleaning be retained in the film surface than macromole, return charging as retention.The main purpose of successfully carrying out the TFF scheme is to optimize TMP and CF, so that can filter the sample of maximum volume, and does not produce the gel that stops up film.If TMP does not have to increase or reduce the average T MP that usually surpasses about 10psi along film length, preferably surpass about 5psi, TMP is " substantial constant " so.As for the TMP level during the whole filtration, keep constant or reduce under higher concentration, to keep selectivity at TMP during the concentration step.Therefore, " TMP of substantial constant " relates to TMP with respect to film length, rather than with respect to filtration time.

Scan

According to a preferred embodiment of the invention, remove Oil globule and casein micelle with predefecation transgenic (" TG ") milk with microfiltration.The transudate of microfiltration wherein keeps lactoprotein by 30kD TFF box system recirculation; Salt and sugar pass through film, and are recycled in the microfiltration retention as diafiltration buffer.The recombinant human alpha-fetoprotein product is present in the clarification Ruzhong that the 30kd film keeps.Now, just in the solution of the complex mixture of milk protein, some of them are very abundant to recombinant human alpha-fetoprotein.Design 100KD protein fractionation step, with the proteinic amount of minimizing infected milk, and preparation is used for the human a-fetoprotein of chromatogram purification.

Before carrying out the 100KD classification, the clarification breast that comprises proteins of interest matter must carry out buffer-exchanged to remove the visible salt in Ruzhong.Therefore, in case finish clarification, then just can be (for example: recombinant human alpha-fetoprotein) use the 20mM phosphate buffer of pH6.5 to use identical 30kD TFF box diafiltration 5 times proteins of interest matter.This preliminary diafiltration is essential, to reduce the salinity in clarification Ruzhong.By reducing salinity, the hydrodynamic radius of recombinant human alpha-fetoprotein increases, and protein is just kept by the HPTFF film of 100kD MWCO easily.Other lactoprotein (except the goat albumin) is not influenced by the mode identical with recombinant human alpha-fetoprotein.Therefore, they can freely pass through the 100kD film, so just are removed and abandon as refuse.

The purpose of 100kD protein fractionation is to remove undesired lactoprotein, lipid and low-molecular-weight pollutant before carrying out chromatograph.Remove pollutant effectively by diafiltration, can reduce the load in the remaining chromatographic step of this method.

Milk is as incoming flow

Embodiment preferred according to the present invention, HPTFF method are used three filter element operations, with clarification from the milk incoming flow, concentrated and classification product.This milk can be the product that comprises the transgene mammal of bio-pharmaceutical or other molecules of interest.In a preferred embodiment, designing this system makes it have high selectivity to molecules of interest.Clarification steps is removed bigger particulate matter from newborn incoming flow, for example Oil globule and casein micelle.Concentrated/classification step is removed most micromolecule, comprises lactose, mineral and water, with purity that increases product and the volume that reduces product.The product of TFF technology is concentrated into proper level subsequently, is beneficial to best downstream purification and whole product stability.This enriched product aseptic filtration that comprises molecules of interest, minimize and strengthen the long-time stability of molecules of interest then to guarantee biological load (being endotoxin).According to a preferred embodiment of the invention, a large amount of degree of purity of production will reach 65% to 85%, can comprise goat antibody (from transgenic goat), lactalbumin (beta lactoglobulin, alpha-lactalbumin and BSA) and low-level residual fat and casein.This partially purified product is the desirable initiation material that is used for conventional downstream chromatographic technique, these downstream technology are further selected and are separated molecules of interest, and described molecule can include but not limited to the recombinant protein of producing in the Ruzhong, immunoglobulin or the fusion rotein of producing in the Ruzhong.

Step 1 (clarification)

Forward Fig. 1 to, utilize the batch-type microfiltration to clarify the transgene mammal breast, breast is preferably from sheep or cattle.Breast is put into head tank, and pump concentrates 2 times (seeing the flow chart of Fig. 1) with newborn retention in loop.In case finish concentrated, then with regard to diafiltration breast retention, so that product and low molecular weight protein, sugar and mineral are by the film in suitable aperture.According to the present invention, design this operation and carry out 2 to 3 hours, and handle 1000 liters of breasts every day.Can amplify technology of the present invention and method, the overall volume of the product that can produce depends on commerce and/or treats the needs of going up specific molecules of interest.

Step 2 (concentrating/classification)

Still be with reference to figure 1, usefulness ultrafiltration (" UF ") concentrates the clarification exudate with classification step one.Clarifying exudate is flowed in the UF head tank, and pump concentrates 2 times with product in loop.Once you begin concentration step is just put into the exudate of UF the newborn retention that step 1 is clarified head tank.Make step 1 and two scale and time allow to operate simultaneously.The exudate of UF comprises small molecular weight protein, sugar and the mineral by film.In case 95% product is accumulated in the retention of UF, promptly stop clarification, begin to concentrate/diafiltration UF material.Product is carried out 5-10 with respect to initial newborn volume doubly concentrate, and buffer is added in the UF head tank.Like this with regard to eccysis most small molecular weight protein, sugar and mineral.Design this operation and carry out 2.5 to 3.5 hours, and can handle every day up to 500 liters of clarification exudates.As mentioned above, can amplify technology of the present invention and method, the overall volume of the product that can produce depends on commerce and/or treats the needs of going up specific molecules of interest.

Step 3 (aseptic filtration)

According to Fig. 1 and the present invention, then clarifying a large amount of concentrate aseptic filtrations.50 to 100 liters of UF retentions of gained are put into head tank, its pump is inhaled by the long-time stability of the absolute 0.2 μ m MF filtration system of dead end to remove most biological load and to strengthen product.The product pump is inhaled by filtration system of the present invention, can directly be filled in the final packaging structure then.In the GMP facility that satisfies clean room standard (as 100 grades of conditions), handle under the condition of molecules of interest.Design this operation and carry out 0.5 to 1 hour, and can handle clarifying a large amount of intermediate every day up to 100L.As mentioned above, can amplify technology of the present invention and method, the overall volume of the product that can produce depends on commerce and/or treats the needs of going up specific molecules of interest.

Embodiment 1

Breast is as the incoming flow of clarification molecules of interest

Following column data provides application of the present invention, and it provides based on the method for film to come from the lactogenesis incoming flow clarification, concentrate and proteins of interest matter that the classification transgenic is produced (for example people recombinate alpha-fetoprotein).According to this embodiment of the invention, the transgene mammal that provides breast to be used to handle is a goat, but also can use other mammal, comprises cattle, rabbit, mice and sheep and pig.

The starting material that has been used for protein fractionation with the microfiltration clarification is reserved a part then and is used for preliminary film optimization research.Design one group and test the effect of estimating in these parameters each, and accurately find out the optimum condition that from the lactoprotein that pollutes, separates recombinant human alpha-fetoprotein.Change a parameter during beginning sometime, until having optimized each parameter, and show the appropriate combination of classification condition.Select the parameter of following scope to be used for the classification experiment:

I. molecular cut off (MWCO) 50-100kD

II. transmembrane pressure (TMP) 5-30psi

III. clarify the pH and ionic strength (20mM phosphate pH6.5) the 0M-1.0M NaCl of breast

IV. clarify emulsion enrichment factor (Cfac) 1X-4X

V. the number 12-20 DV ' s of diafiltration volume

VI. clarification newborn batch (seeing material)

VII. film restores (seeing the VII joint)

With the TFF system with 0.1M NaOH sterilization, with the flushing of USP water, and with the 20mM sodium phosphate buffer balance of pH6.5.Measure and write down initial water permeability ratio.During beginning 4 liters of clarification breasts are concentrated certain multiple and volume is reduced to 1 liter.Newborn with this spissated clarification of 20mM sodium phosphate buffer diafiltration of pH6.5 then.Should the breast diafiltration be 4.0 to the O.D. of 280nm place, rather than with the diafiltration volume of its diafiltration fixed amount.This light absorption roughly is equivalent to the total protein of 6g/l.Select this total protein concentration be since subsequently chromatographic step to the restriction of this technology.In case after finishing diafiltration, just this system is drained and washes, then phosphate buffer and final retention merging with 1 liter of phosphate buffer.Use the recombinant human alpha-fetoprotein after the 0.2 μ m bag type filter aseptic filtration classification then.

Analyze total protein, AFP concentration and the contaminating protein of the recombinant human alpha-fetoprotein after the classification then with RPC.In addition, run sds gel and further estimate remaining contaminating protein.

HPTFF of the present invention system is made up of Pall Centramate four dosimeters with 2 peristaltic pumps.First pump is used for the recirculation of retention, and second pump is used for the recirculation of exudate.This pump suction scheme is known as common stream and flows (Co-Current flow), referring to Fig. 1 and 2.It is generally used for the complete trails length balance TMP along film most, and is more even to guarantee classification.

The result

I. retaining molecular weight (MWCO)

II. transmembrane pressure (TMP)

The exploitation of protein fractionation step is included in the recirculation mode carries out process optimization with 100KD HPTFF.Come the characterizing process condition by comparing TMP specific discharge (as follows) during beginning.Parent material is the clarification breast with the 20mM phosphate buffer diafiltration of pH6.5.Buffer conditions is fit to this classification, because low salt concn increases the reservation of rhAFP molecule.This optimization shows that our best TMP should be between 12 to 20psi, and it is outside the film control zone and enter film by the transition region of hierarchical control.The cross-flow flow velocity is not further optimized, but remains on the 0.6L/ branch/ft2 of manufacturer recommendation.

In case finish optimization, just can determine that a cover operational factor is used for the HPTFF classification.These are identified for the operational factor of protein fractionation, and technology is critical for keeping repeatably.Need the key parameter of monitoring to comprise transmembrane pressure, cross-flow velocity and buffer conditions.

III. clarify emulsion pH and ionic strength

IV. clarify the enrichment factor (CFac) of breast

This protein fractionation is concentrated to reduce volume and to save the required amount of buffer of diafiltration and form by initial 4X.Do not use concentration step when beginning during the process exploitation, but proved afterwards that this was necessary, because too many with the buffer volume of 1X diafiltration needs.Then the 10-20 times of volume of clarification breast diafiltration after concentrating.

With the 1X classification

The clarifying breast of the preliminary diafiltration of 20mM sodium phosphate buffer with pH6.5.Owing to removed contaminating protein from retention, Once you begin the diafiltration flux just begins to raise.

TMP	12psi
		Cross-flow	0.6L/min/ft2
Retention pressure falls	15-20psi
		Exudate pressure falls	15-20psi-is mobile altogether
Typical case's flux	80-100LMH

Table 3.HPTFF operational factor

With the 4X classification

The clarification breast is tentatively concentrated certain multiple, and volume is reduced to 25% of initial volume.The initial decline of flux can be referring to the DV0 point of this figure between diakinesis.This spissated clarification breast of the 20mM sodium phosphate buffer diafiltration of usefulness pH6.5 is 20 times then.Owing to removed contaminating protein from retention, Once you begin the diafiltration flux just begins to raise.From the TFF system, wash final 4X concentrate with isopyknic buffer then, final concentrate is reduced to 2 times to initial volume.

Table 5.TFF operational factor

TMP	12psi
		Cross-flow	0.6L/min/ft2
Retention pressure falls	15-20psi
		Exudate pressure falls	15-20psi-is mobile altogether
Typical case's flux	80-100LMH

V. diafiltration volume number

A replacement scheme with the diafiltration volume fractional defecation breast of fixed number is the O.D. that diafiltration to retention is reduced to the 280nm place.Determine to stop the accurate point of diafiltration in the light absorption of 280nm by retention.The target absorbance is 4.0AU in 4X concentrates.280nm light absorption 4.0 roughly is equivalent to the 6mg/ml total protein with the RPC analysis.No matter this makes initial concentration how, and this technology can both produce the classification product of identical total protein concentration consistently.After with the dilution in 1: 1 of 20mM phosphoric acid buffer flushing liquor, the goal setting of final total protein concentration is 2.8-3.2mg/ml.

This figure has shown that immediately the diafiltration volume is to flux and the diafiltration trend to light absorption.Exponential decay is roughly followed in light absorption when diafiltration is carried out.In case removed the protein that great majority pass through, remaining protein has just constituted final classification product.This end product typically mainly comprises recombinant human alpha-fetoprotein, albumin and casein.

Table 6. percentage yield (according to RPC)

Sample	Concentrate hAFP (mg/ml)	Gross protein (mg/ml)
			201-TR-0001A sample #3	0.61	N/A
201-TR-0001A sample #7	0.94	2.98

Each sample is prepared two dilutions and retest.Concentration is reported as the meansigma methods of four injections.

RPC analyzes and shows the important data of two classes, recombinant human alpha-fetoprotein concentration and total protein concentration.Last table is presented at classification begin before the initial concentration of recombinant human alpha-fetoprotein be 0.61mg/ml.After the classification, ultimate density is 0.94mg/ml under 2 times enrichment factor.The hypothetical target enrichment factor is 2X, can calculate ultimate yield with the following methods.

% yield=((ultimate density/enrichment factor)/initial concentration) * 100

% yield=((0.94mg/ml/2)/0.61mg/ml) * 100=78% yield

Annotate: this yield only is roughly to be worth, because used enrichment factor only is a desired value

Table 7. gross protein (according to RPC)

Sample	Concentrate hAFP (mg/ml)	Gross protein (mg/ml)
			201-TR-0001A sample #3	0.61	N/A
201-TR-0001A sample #7	0.94	2.98

Each sample is prepared two dilutions and retest.Concentration is reported as the meansigma methods of four injections.

Measure the total protein concentration of final recombinant human alpha-fetoprotein sample with rpc.This is the information that will be used for the post application of sample subsequently.Concentrate down at 4X, the target total protein concentration of final classification recombinant human alpha-fetoprotein tentatively is decided to be 6mg/ml.Dilute fractionated recombinant human alpha-fetoprotein with isopyknic dcq buffer liquid then, make that the final objective total protein concentration is 3.0mg/ml.

Lipidated protein (according to RPC and SDS PAGE)

Analyze the contaminating protein of fractionated recombinant human alpha-fetoprotein then with RPC.In addition, carry out SDS PAGE and further estimate remaining contaminating protein.

Lipidated protein (according to RPC and SDS PAGE)

Above RPC chromatogram helps to prove the protein fractionation that is undertaken by 100KD slipstream film.First RPC chromatogram shows that 1.0mg/ml recombinant human alpha-fetoprotein purification is with reference to the elution time with 4.2 minutes.Second RPC chromatogram is presented at the preceding clarifying initial breast of protein fractionation.The 3rd RPC chromatogram shows fractionated recombinant human alpha-fetoprotein (No. 6 peaks) and remaining infected milk protein.1 and No. 2 peak is unidentified lactoprotein, and 3 and No. 5 the peak is a casein, and No. 4 peaks are sheep blood serum albumin, and No. 7 peaks are high molecular lactoproteins.The difference of second and the 3rd chromatogram shows the relative quantity of the contaminating protein matter of being removed by the protein fractionation step.

VI. film recovers

Standardized water penetration (NWP) is evaluated for film performance measuring from recovery in service.If do not measure the means of film cleaning effectiveness, film properties will be lost with membrane lifetime.Measure the NWP of new film, and as all further reference points of the NWP data of record.When recover original NWP 90% when above, think that typically this film is " cleaning ".Figure below is presented at the trend of a kind of Pall OS100C12 box that uses between development period.

Effective way that show to use not the suitably effect of the box of cleaning is that the film with obstruction carries out this technology practically.Figure below shows that film NWP does not recover to surpass 60% o'clock of former NWP, 3 isolating operating technology flux.Operation 081403A, 081403B, 081503A and 081503B show that the technology flux is in the 50-70LMH scope.After these four operations are finished, at 50 ℃ with 0.5M NaOH and the thorough cleaning film of 400ppm NaOCl.Measuring N WP shows to have returned to more than 90% of former NWP.Operation 081803A, 081803B, 081903A show that the technology flux is in the 110-130LMH scope.The technology flux of the new film that these technology flux and operation 082003A are indicated is corresponding.

With SDS Page the final classification product of each run is compared.Although the technology flux significantly reduces when with the obstruction film, classification does not demonstrate and is subjected to very big influence.It is effectively also can be used for this grading technology that these data support previously described NWP to recover model.

According to the present invention, the target of isolating proteins of interest matter with the HPTFF method from contaminating protein is confirmed.This fractionated purpose is to keep target protein (AFP) and filter out most infected milk protein.RPC, total protein and sds gel result show that finally the proteinic concentration of infected milk can effectively reduce.Use 100KD slipstream film of the present invention and method to make the whole concentration except that recombinant human alpha-fetoprotein and CSA all be lowered.

Retaining molecular weight (MWCO)

Membrane aperture and chemical property have been brought into play important effect in the classification effectiveness.Estimate several MWCO films, comprise 30KD, 50KD, 70KD and 100KD.If MWCO is too low for example is 30KD, no matter uses what operating condition, and contaminating protein all can be retained.Use the classification in this aperture not go out contaminating protein from the recombinant human alpha-fetoprotein classification effectively.Estimate every kind of more wide-aperture retention properties and selectivity.30kd, 50kd and 70kd are proved to be and can keep many infected milk albumen, and can not be used for this classification effectively.The 100kd film has shown any substantial loss of product when initial before optimization.The aperture of this film is proved to be available maximum diameter of hole, yet still can keep recombinant human alpha-fetoprotein.

Various types of films comprise regenerated cellulose, polyacrylonitrile (PAN) and modified poly (ether-sulfone) (PES).Every kind of film all has the properties of combination of it self uniqueness, and can influence classification.In case select suitable membrane aperture, just estimate the type or the chemical property of this film.Since its uniform aperture and neutral membrane charge, the modified poly (ether-sulfone) (PES) of selection Pall Corp.Omega.

Transmembrane pressure (TMP)

In recirculation mode, determine best transmembrane pressure with 100KD HPTFF film.Parent material is the clarification breast with the 20mM phosphate buffer diafiltration of pH6.5.This buffer conditions is fit to this classification, because low salt concn increases the reservation of rhAFP molecule.Optimize curve and show that technology TMP should be about 12-20psi, enters transition region just.In the film control zone, lower TMP has amplified the charge interaction between infected milk albumen and the film, has reduced their transmission.In the gel layer control zone, higher TMP orders about all proteins to the surface, has increased the mass transfer coefficient of bulk solution.This makes recombinant human alpha-fetoprotein reclaim reduction and fractionated efficient reduces.Further do not optimize the cross-flow flow velocity, but remain on 0.6L/ branch/ft2 that manufacturer is recommended.

The pH and the ionic strength of clarification breast

The isoelectric point, IP of recombinant human alpha-fetoprotein is about 5.0, and the isoelectric point, IP of film is about 7.0.In order to make the reservation maximization of recombinant human alpha-fetoprotein, selecting the pH of buffer solution is 6.0-6.5.Under these conditions, film and recombinant human alpha-fetoprotein molecule all have negative charge and repel mutually.Ionic strength is being brought into play significantly effect in the retention properties of recombinant human alpha-fetoprotein molecule.Under the condition that improves salt condition (＞1.0ml NaCl) under most of operating conditions molecule can be more freely hole by the 100kd film.Under the condition that the salt condition reduces, situation is just in time opposite; Most recombinant human alpha-fetoprotein molecules are retained.Known have two kinds of factors that this phenomenon is had contribution.The reduction of salinity has increased the charge effect between protein and the film.In addition, the reduction of salinity has caused recombinant human alpha-fetoprotein " swelling " effect of water layer on every side.The effect of these factors combine has caused this molecule to be kept by the 100KD film, and really not so under the normal condition.

Clarify newborn enrichment factor (Cfac)

The enrichment factor of clarification breast is 2 times of full milk concentration when initial.Then should the clarification breast with 100KD HPTFF film system level of the present invention.Typically, diafiltration (C _D) optimum be by maximum gel layer concentration (C _G) multiply by C _GThe value of/e is determined:

C _D＝C _G/e＝0.37C _G

Be used for the optium concentration of fractionated diafiltration part and be not easy calculating, because the protein of significant quantity is removed when the clarification breast is concentrated.Therefore CG is always changing, and as semilog diagram predict the concentration of overall protein do not increase.

Therefore reach the optimum of diafiltration according to experiment.Come this clarification breast of diafiltration with 1X, 2X and 4X.The effect that increases enrichment factor before classification has increased and has reached the required diafiltration volume number of same purity level.Yet,, can realize that still the buffer demand reduces by 50% even the spissated benefit of 4X is that the diafiltration volume is double.

Diafiltration volume number

The diafiltration volume number that is used for the classification recombinant human alpha-fetoprotein is less than the some decision of the amount of the recombinant human alpha-fetoprotein that is lost in exudate by the amount of the contaminating protein of removing.This point can be estimated by observing SDS PAGE information.It is about 10DV ' s that 1X concentrates required preliminary diafiltration volume number, and it then is 20DV ' s that 4X concentrates.The most effective scheme is verified to be the latter, and promptly 4X concentrates 20DV ' s down.

Select to change the diafiltration terminal point in preliminary Q-post exploitation back.The amount of monitoring total protein between operation, this is proved to be the mode that right quantity diafiltration volume is used in more effective prediction.

Being selected to fractionated final method is that diafiltration to retention is reduced to below a certain O.D. of 280nm mensuration.Determine to stop the accurate point of diafiltration according to the light absorption of 280nm place retention.When concentrating, 4X finds that target light is absorbed as 4.0AU.4.0 light absorption at the 280nm place roughly are equivalent to the 6mg/ml total protein with the RPC analysis.No matter this makes initial concentration how, and this method can both produce the classification product of identical total protein concentration consistently.The selection of this total protein concentration be since subsequently anion-exchange chromatography step to the restriction of technology.Contaminating protein influences the load of Q-post to the ratio regular meeting of AFP, so must be consistent between operation.After with the dilution in 1: 1 of 20mM phosphoric acid buffer flushing liquor, the final total protein aimed concn of Q-load is set at 2.8-3.2mg/ml.

Newborn batch of clarification

Be used for the batch influential of fractionated clarification breast for the infiltration capacity that reaches same endpoint (O.D.@280nm) needs.Early stage clarification breast of collecting of lactogenic and batch comparing of when lactogenic finishes, collecting, more than the amount of total protein 50%.Yet the clarification batch in the product amount tend to keep constant.The difference of protein concentration may be because higher in the proteic amount of the early stage infected milk of lactogenic.The breast in these clarification breasts of early stage batch and late period batch is compared and is correspondingly needed many diafiltrations about 50%.With comprising that the analysis that RPC, SDS PAGE and Bradford total protein are measured all shows,, all has similar result between all classifications batch no matter use which kind of clear material.

Film recovery-standardized water penetration (NWP)

HPTFF box system is designed to reuse during reaching 1 year.Therefore, it is important cleaning film later on effectively at each run.The cleaning solution that is used for the 100KD film is 40-50 ℃ 0.5M NaOH and a 400ppm bleach, washes 1 hour.This cleaning program is proved to be can the standardization of efficient recovery film water penetration (NWP).If former NWP when adopting new box 90% within, just think the NMP " recovery " of film.Period shows the curve chart of NMP, and film should have and moves 60 times life expectancy at least.

For proving that further protein fractionation is reliable and film recovers to predict film properties, deliberately stop up this film, be used for protein fractionation then three times.The technology flux is about half of corresponding cleaning film, but astoundingly, classification efficiency does not change.Shown concordance with the material after RPC and the SDS PAGE analysis classification.These data show that water flux can be well under recovering 90% of its regulation.

The data that obtain from this experiment show, carry out protein fractionation with 100KD MWCO HPTFF of the present invention system, can reduce the proteic concentration of infected milk effectively.This technology can be moved when liquid flux is 80 to 100 LMH effectively, when the upper reaches purification process with other needs MIN equipment investment when for example chromatography compares.The relative purity of clarification Ruzhong recombinant human alpha-fetoprotein is 6-10% when initial, rises to 30% purity after classified.The yield of this technology continues to remain on 80% scope, and is suitable with subsequently purification step.

According to the present invention, experimental strategy is the relation between the filtering technique variable of determining to control on a large scale (CM, V, TMP, T), and wherein V is a flow velocity, for example can control product by, reservation and quality.Determine its relation by a series of independent laboratory scale experiments, and determined the optimal operations window.These optimal parameters are combined in the serial experiment, study total recovery and mass balance.Determine performance by product yield, clarity and flux efficient.In independent laboratory scale serial experiment, investigated following state-variable.

Concentrate (Cm)Determine best newborn enrichment factor with experience part product by data.Product flux (Jp) is illustrated in set time interior every square metre product percent of pass.The product flux will be measured according to the relation of the enrichment factor during the clarification steps (operating unit #1).

Still with reference to figure 1, followingly provide explaining to key element of the present invention.

Fig. 1 key element

The technological process explanation

Flow process number explanation

The original transgenic breast of 1a

1b microfiltration CIP solution

The microfiltration retention of discharging after the 2a diafiltration

CIP solution after the use that 2b discharges

MF retention (loop) in 3 technologies

4 MF CIP recirculatioies (loop)

5 microfiltration leach thing

6 ultrafiltration CIP solution

CIP solution after 7 uses of discharging

8 ultrafiltration chargings (microfiltration leaches thing)

UF retention (loop) in 9 technologies

10 ultrafiltration exudates (being used for diafiltration MF retention)

11 spissated clarification breasts

12 UF CIP recirculatioies (loop)

13 AF CIP solution

14 sterile filters chargings

The concentrating clarifying breast that 15 biological loads reduce

CIP solution after 16 uses of discharging

With regard to its most extensive aspect, the high performance tangential flow filtration technology that the present invention paid close attention to provided herein be included under some TMP and the flux condition mixture with material to be separated in device that is designed for the HPTFF type system or module by one or more filter membrane.TMP is remained in the scope of flux to the pressure dependence district of TMP curve, promptly in the scope that is not more than transition point TMP value.Therefore, carrying out filtering flux range is at about 5% to 100% of transition point flux.See Figure A and B wherein describe flux to the TMP curve along transition point.Therefore, film selective retention substances of interest is as retention, and less material passes through film as leaching thing, and perhaps substances of interest is as leaching thing reservation of the pollutant tunicle in the mixture by film.Should be noted in the discussion above that for the substances of interest of ultrafiltration preferably molecular weight at least about 1000 daltonian biomacromolecules, most preferably polypeptide and protein.Also preferred substances of interest ratio will isolating with it material be that pollutant are greatly less than 10 times, perhaps than will isolating with it material little of 10 times.

The statement of Shi Yonging herein " will leach thing by the means that leach thing chamber recirculation parallel with flow direction in the filter chamber " and represent a kind of mechanism or device, the segment fluid flow that its guidance leaches the thing chamber flows, and its flow direction extremely exports by adjacent filter chamber flow direction parallel and basic identical (allowing to occur some whirlpools) from filter chamber's inlet with fluid.Preferably, these means are that pump is inhaled means.

Should be noted in the discussion above that TMP does not increase with grading time, and during filtering, needn't keep constant.TMP can keep constant or also can carry out and reduce with filtration.If concentrate retention, so preferably in the process of concentration step, reduce TMP.

Every kind of film preferably have can keep particle diameter up to about 10 microns, more preferably 1KDa to 10 micron the aperture of material.Can comprise protein, polypeptide, colloid, immunoglobulin, fusion rotein, immunoglobulin fragment, mycoplasma, endotoxin, virus, aminoacid, DNA, RNA and carbohydrate with the examples of substances of ultra-filtration and separation.Can comprise mammalian cell and microorganism, for example antibacterial with the isolating examples of substances of microfiltration.

Because film filter no all roses, the reservation molecule that has enough some expectations of ambassador of some holes or defective filters, therefore a preferred aspect is to utilize the film that surpasses a same apertures herein, wherein places this film so that it is parallel to each other stacked, and preferred one deck is on another layer.The quantity that is preferred for the film of this purpose is 2.

Although the suitable flux range that keep-ups pressure in above-mentioned technology is about 5 to 100%, flux is low more, and the film surface area that needs is just big more.Therefore, in order to make the cost minimum of film, preferably make the higher-end of flux at spectrogram at operating pressure.Preferred range is the about 50 to 100% of transfer point flux, and preferred scope is about 75 to 100% of a transfer point flux.

Although do not need to keep the TMP substantially constant, preferably keep the TMP substantially constant along the film surface.Such condition can realize by the thing side build-up pressure gradient that leaches at film usually.Therefore, leach the thing recirculation according to direction identical or parallel direction and leach the thing compartment by this device with mix flow in the retention compartment of defecator.Import and the outlet pressure of regulating recycled materials fall in the pressure of striding the retention compartment so that stride the pressure degradation that leaches the thing compartment.

Several practical means can be used for realizing that this leaches the thing barometric gradient.Some examples of preferred embodiment are structures as shown in accompanying drawing 2A and 2B.According to these structures, solute to be separated is by inlet tube 36 accesss to plant, if product to be separated in fermentation liquid, inlet tube 36 is communicated with the fermentation tank (not shown) so.It can also with preserve transgenic (Tg) breast or in the harvesting culture systems the later cell lysate of cell or the container (not shown) of supernatant be communicated with.Flow velocity by pump means 40 adjustable pipes 36.Pump can be the pump of any appropriate well known by persons skilled in the art, and can regulate flow velocity according to filtering property well known by persons skilled in the art.

In microfiltration of the present invention unit 30, optional working pressure meter 45 is measured from pump means 40 mobile inlet pressures.Fluid in the inlet tube 36 enters filter element 50.This filter element 50 is included in its filter chamber that enters the top 51 and leaches thing chamber 52 in exit portion.These two compartments with filter membrane 55 separately.The mobile direction of inlet fluid is parallel to the filter membrane 55 in the filter chamber 51.Filter chamber 51 accepts to comprise solute in the upper end mixture wherein contains molecules of interest.The molecular energy littler than target molecule enters by film 55 and leaches thing or discharge chamber 52.Spissated retention passes through through outlet 60 from filter element 50, if necessary can use microfiltration (MF) film 65 to collect and further this retention of handling, and to obtain the substances of interest of expectation, comprises by other film.During whole process, and for the purpose of quality control, the present invention also comprises a series of sample spot 99, to allow monitoring molecular concentration, pH and pollution-" path B ".As an alternative, retentate stream can loop back jar or the fermentation tank 35 " path A " that produces mixture, is recycled to be further purified by inlet tube 36.

Enter the solution that comprises molecules of interest that leaches thing chamber 52 by film 55 and also can leave filter element 50 by the outlet 70 in filter element 50 same end, the retention fluid then flows out through outlet 60.But the solution and the molecules of interest that flow through outlet 70 are sent back in the jar 35, and measure with further processing with piezometer 72.

Similarly, shown in Fig. 2 B, also consider system according to dual TFF of the present invention.

In the structure shown in Fig. 2 A, film need be placed with respect to chamber 51 and 52 provides specified flow velocity and transmembrane pressure poor.The film that is used for technology of the present invention generally is tubulose and other structure of plain film shape, coiling lamellar, cylindric, concentric circular tubular, varying cross-section, its single assembling or assembling in groups, and in filter element serial or parallel connection.General this device that makes up makes to filter and leach the thing chamber to operate on the film length.

Suitable film can not have blockage problem basically not expecting separating substances to open in expectation material and the mixture and is in the speed that is enough to make system's continued operation.The example comprises that the aperture typically is 0.1 to 10 micron microporous membrane, and can be so that it keeps all granules bigger than specified diameter.The film that is used for microfiltration applications and TFF application according to the present invention preferably all is ceramic.Ultrafilter membrane has littler hole, and characterizes according to the proteinic size that will keep.Can obtain from 1000 to 1,000, the film that 000 dalton name molecular weight limit increases gradually.

Ultrafilter membrane is the most common to be applicable in the technology of the present invention.Ultrafilter membrane generally is asymmetrical, has thin film or the skin of being responsible for its separating power on its upstream face.These films are normally made by regenerated cellulose or polysulfones.

The film filter of tangential flow filtration system 80 can according to pending liquid volume with provide as heteroid unit with multiple different apertures.In relatively large scope, being specially adapted to of the present invention is those known, commercial available tangential flow filtration unit.

In a substituting and preferred device, and because aforesaid reason, the microfiltration unit 30 of accompanying drawing 2A comprises a plurality of, preferred two filter membranes, as film 56 and 57.These films are stacked according to parallel construction.

The present invention also comprises plural serial stage technology, and wherein the thing that leaches from above-mentioned technology passes through in second tangential flow filtration device than the littler filter membrane of membrane aperture in first device, and this second filtering thing recirculation that leaches returns in first device, and repeats this technology.

Be applicable to technology of the present invention or unite a kind of slipstream system 80 of use shown in accompanying drawing 2B with microfiltration unit 30.At this, first container 85 links to each other with the filter chamber 96 that is arranged in filter element 95 through inlet tube 90.The first front pump means 100 are between first container 85 and filter chamber 96.Filter chamber 96 links to each other with first container 85 through outlet 110.In filter element 95, filter chamber 96 by first filter membrane 115 be located immediately at first under it and leach thing chamber 97 and separate.First leaches thing chamber 97 has outlet 98, and its inlet with chamber 97 links to each other, and have be positioned at pipe 98 leach thing pump means 120.The pipe 45 that links to each other with outlet 98 also links to each other with second container 120.

This container 120 links to each other with second filter chamber 127 that is arranged in second filter element 130 through inlet tube 125.The second front pump means 133 are between second container 120 and filter chamber 127.In filter element 130, filter chamber 127 by second filter membrane 128 be located immediately at second under it and leach thing chamber 129 and separate.Second leaches thing chamber 129 has outlet 135, and its import with chamber 129 links to each other, and has the filter pump means 140 that are arranged in pipe 135.The pipe 1255 that links to each other with outlet 135 also links to each other with the 3rd container 150.

This container 150 links to each other with the 3rd filter chamber 157 that is arranged in the 3rd filter element 160 through inlet tube 155.The 3rd front pump means 165 are between the 3rd container 150 and filter chamber 157.In filter element 160, filter chamber 157 by the 3rd filter membrane 165 be located immediately at the 3rd under it and leach thing chamber 159 and separate.The 3rd leaches thing chamber 159 has and pipe 155 outlets that link to each other 170, and it links to each other with first container 150, is recycled in this former jar so that leach thing.Also sampling point 99 and piezometer 175 are to monitor this technology.

Technology of the present invention is highly suitable for commercial size.It can according to batch or continued operation, perhaps move in semicontinuous mode, for example with the continuous-flow that comprises the solution of expecting material serve as the basis by the slipstream filter, until whole large quantities of materials thereby be filtered, between filtration stage, can insert cleaning step.Handle new a collection of solution then.In this way, can carry out continuous circular treatment, in the relatively short time, obtain the acceptable a large amount of expectation products of purity.

The specific characteristic of tangential flow filtration described herein, and it provides continuous filtration to contain solid solution and do not have the ability that filter stops up, cause a kind of very advantageously can continuous foundation and commercial size being used to of using separate technology with the purifying biological product.In addition, this technology can be used for the biomolecule of wide region, for example protein, antibody, cell fragment and the cell culture lysate in transgenic source.

Following examples are used to further specify the present invention, rather than limit.The disclosure of all lists of references of quoting in these embodiments, is all clearly incorporated this paper into.

The clarification module

Can be used for film of the present invention and can manufacture the production module with several forms.The most common form that is used for tangential flow filtration is:

-flat board

-screw winding

-doughnut

In these modules every kind basic stream as shown in Figure 3, it illustrates the fluid flowing path of incoming flow by different HPTFF and TFF module.

Charging and/or leach thing passage interpolation and go into web plate (screen) in be everlasting screw winding and flat sheet mould is to increase the turbulent flow in the passage and to reduce concentration polarization.In hollow fiber module, then there is not this selection.Promote turbulent passage under low cross-flow rate, to have the higher quality carry-over factor, mean under the lower situation of the demand that pumps into and realized higher flux.Therefore promote that turbulent feeding-passage is more efficient than open channel.In flat sheet mould, use the suspension web plate can bring some benefits of open channel and promotion turbulence channel.Accompanying drawing 7 has shown dissimilar channel designs.

Dull and stereotyped

(often being called box) in the Flat Membrane module, the number tunics or with or do not have the separate mesh flaggy of alternately arranging and be stacked, then it is sealed in the packing.Feed fluid is pumped in the alternate channel of an end that piles up, and leaches thing and enters by film and leach in the thing passage.Flat sheet mould has higher packed density (membrane area of every base area) usually, allows linear amplification, and some of them provide open or promoted the selection of turbulence channel.

Screw winding

In the screw winding module, the intercooler core of alternative film and separate mesh flaggy coiling hollow.Incoming flow is pumped to an end and flows downward along spool axis.Leach thing and turn around by film and with respect to core, it is removed at this.Separating web plate increases turbulent flow in the stream, and it is become than doughnut module more efficiently.A shortcoming of screw winding module is, they cannot linear amplification, because charging flow path length (reel length) or leach logistics road length (reel width) and must change in proportion.

Doughnut

Hollow fiber module is made up of pencil film pipe, and the film pipe has narrow diameter, typically in 0.1 to 2.0mm scope.In hollow fiber module, incoming flow is pumped in the inner chamber (inside) of pipe, leaches thing and arrives shell-side by film, is removed this its.Because the charging stream is very open, even only produce than low-shearing force in medium cross-flow rate.Although this is for useful to the extremely sensitive product of shearing force, in general because it needs very high pump capacity to realize that competitive flux will reduce the efficient of module.

For the experiment finished of useful microfiltration systems, except charging-and-flow out experiment, the equipment of use is as follows:

The 60lpm pump is to associated pump calibration (pump curve)

1 " the clean pipe of OD rustless steel

0.2sqft or the 0.2 μ m aperture ceramic membrane of 1.5sqft

" the clean membrane support of the rustless steel of outlet that has one 1/2

1/4 " ID is flexible oozes out property management

Diaphragm valve on the retention line

2 piezometers

Steel 1.2 L charging reservoirs

3/4 " the flexible reservation property management of ID.

For all HPTFF experiments, cooperate aforementioned device with following equipment:

The diaphragm valve of maximum output 800mLPM

1/4 " the flexible pressure pipe of ID on wired

1 piezometer that is used to measure feed pressure

2 diaphragm valves on retention and exudate line

0.2sqft or the 30kDa NMWCO PES Pall Filtron Centramate film of 1sqft

Rustless steel Pall Filtron Centramate membrane support

1 rustless steel u-shape bend pipe that links to each other with the exudate mouth.

Film is selected

The film that is selected to HPTFF of the present invention system is selected from the film with different geometries and nominal molecular cut off.Previous research is for using the high MWCO UF film and the ceramic membrane of polymer-matrix to explore in clarification steps.Breast is concentrated 2X downwards, carry out HPTFF then and test all films.Test them by multiple operation and cleaning then, analyze the reusability of these films.When standardized water flux maintains 80% when above of new film, think that promptly this film is resumed, and can be used for handling next time.Plain film polymer bellows all can not be kept and recover the target water flux after using 3 times, and ceramic membrane then can recover above 60 times.This is because can enough higher chemical concentrations and more pyritous more harsh conditions cleaning ceramic.After the circulation above 20, the 30kDa ultrafilter membrane still can be kept and recover high water flux.

Test the first module that is used to clarify emulsion and passes through proteins of interest matter with 0.2 μ m name ceramic tubular film.Test second system that is used to catch molecules of interest with the plain film ultrafilter membrane of 30kDa molecular cut off.

Analytical method

Analyze from the recombinant human alpha-fetoprotein in the sample in each laboratory sample (rhAFP) content by a-protein HPLC, analyze its degraded by SDS-PAGE, (IEF) analyzes its modification by isoelectric focusing, and (SEC) analyzes its gathering by size exclusion chromatograph.

Operation

Use the ceramic micro filter film of 0.2 μ m molecular cut off to carry out a series of control experiment, wish to understand the technological operation relation.Measurement products flux (Jp) is because it is relevant with newborn concentration (c) with flow velocity (u), transmembrane pressure (TMP), temperature (t).In case determined relation, just can determine the optimal operations window, and test amended technology.Collected specimens, and collection mass balance data are used to analyze initial product yield and flux scale.(seeing also accompanying drawing 2A and 2B).

Temperature experiment

Its objective is the scope of determining operative temperature, this operative temperature obtains with the best rhAFP flux of lowest volume by 0.2 μ m, 3mm passage ceramic membrane.In order to degrade with SDS-PAGE and Western engram analysis rhAFP, before merging breast, measure the pH of each several part breast at the technology run duration.Breast is integrated with the MF head tank, the record cumulative volume.At this moment, the MF pump controller is heightened 45Hz (assigning to about 20L from about 5L/) from 20Hz.Write down all parameters of each continuous time point, for example temperature, pressure, cross-flow flow velocity, exudate flow velocity and volume.This MF loop of operation is 5 minutes in recirculation (path A), and transmembrane pressure is adjusted to 12psig and recirculation (path A) 5 minutes (temperature maintenance is at 20 ℃).The exudate line points to discharges, and is concentrated (collection exudate) with respect to original newborn volume by 2X until breast.Temperature maintenance is at 20 ℃.Sample thief 2 and 3 from charging reservoir and exudate line.Ooze out then among the line return path A, and recirculation 10 minutes.Sample thief 4 and 5.Temperature is risen to 25 ℃.Then system's recirculation 10 minutes, sample thief 6 and 7.Temperature is risen to 30 ℃.Then system's recirculation 10 minutes, sample thief 8 and 9.Temperature is risen to 35 ℃.Then system's recirculation 10 minutes, sample thief 10 and 11.Temperature is risen to 40 ℃.Then system's recirculation 10 minutes, sample thief 12 and 13.Close pump then, sample preservation at 2-8 ℃ and send to quantitatively.Use the IEF analytic sample.

The MF breast concentrates experiment

According to a preferred embodiment of the invention, the purpose of this experiment is to determine initial newborn concentration range, and this initial concentration obtains with the best flux of lowest volume by the proteins of interest matter of 0.2 μ m, 3mm passage pottery MF film.

In order to operate, before merging breast, measure the pH of each several part breast.Breast is integrated with the MF head tank, the record cumulative volume.At this moment, the MF pump controller is heightened 45Hz (assigning to about 20L from about 5L/) from 20Hz.Write down all parameters of each continuous time point, for example temperature, pressure, cross-flow flow velocity, exudate flow velocity and volume.This MF loop of operation is 5 minutes in recirculation (path A), and transmembrane pressure is adjusted to 12psig, and recirculation (path A) 5 minutes (temperature maintenance is at 20 ℃).Transmembrane pressure is adjusted to 15psig, and recirculation (path A) 5 minutes.The exudate line points to discharges, and is concentrated until breast, collects the 550ml exudate, then exudate line return path A.(recirculation 10 minutes). Sample thief 2 and 3 from charging reservoir and exudate line respectively.

The exudate line is used for path B, in the charging reservoir, adds the 600ml breast.The exudate line points to discharges, and is concentrated until breast, collects the 500ml exudate, then exudate line return path A.(recirculation 10 minutes).Sample thief 4 and 5 from charging reservoir and exudate line respectively.Then the exudate line is pointed to path B, in the charging reservoir, add the 500ml breast.The exudate line points to discharges, and is concentrated until breast, collects the 500ml exudate, then exudate line return path A.(recirculation 10 minutes).Sample thief 6 and 7 from charging reservoir and exudate line respectively.Then the exudate line is pointed to path B, in the charging reservoir, add the 380ml breast.The exudate line points to discharges, and is concentrated until breast, collects the 400ml exudate, then exudate line return path A.(recirculation 10 minutes).Sample thief 8 and 9 from charging reservoir and exudate line respectively.Close pump then, sample preservation at 2-8 ℃, is used for quantitative proteins of interest matter, analyze its degraded and gathering, analyze gathering and analyze the isoelectric point, IP migration with IEF with SEC by a-protein analysis, SDS-PAGE and Western.

By from lactogenesis, removing particulate matter for example fat, casein micelle and antibacterial, use the technology of HPTFF as clarification and stable rhAFP in newborn substrate.In biotechnology and dairy industry, use HPTFF to remove impurity and enriched product with limited mode.According to the present invention, in order effectively to use HPTFF, importantly select suitable film, optimize technological parameter (temperature, transmembrane pressure, cross-flow flow velocity and newborn concentration) to obtain high product flux, exploitation is cleaned and store method guarantees that the useful life of film is longer.The basic parameter of experiment and is applied in the transgenic goat milk to verify aforesaid operating parameter according to the present invention as described here.The cleaning and the preservation condition of film have also been studied.After finishing HPTFF technology, also developed the aseptic filtration step to remove remaining any antibacterial from the clarification Ruzhong that contains proteins of interest matter.Then technique information is diverted on the pilot plant, is carried out preliminary engineering operation thereon.Some technological design standards comprise, do not use additive in order to avoid need water for injection, film operating period limit for length, high yield and processing time short.Optimal process of the present invention is designed to scalable be used for pilot scale and production operation.

The non-transgenic charging-and-flow out and test

Analyze with the liquid flux decay of 0.2 μ m ceramic micro filter film between diakinesis, because the supply of available non-transgenic breast is very sufficient with the non-transgenic breast.The equipment that is used for this experiment comprises with microfiltration tests described identical equipment, but also needs to increase by second charging reservoir and the feed pump, so that emulsion flows in the charging reservoir of microfiltration systems to flow out the identical speed of film with exudate.The equipment sketch map is:

From figure A as seen, fill the charging reservoir with the 1500ml breast, with the 45Hz primer pump.This system of operation is 10 minutes in recirculation, and does not apply retention pressure.Write down all parameters.Then retention pressure is increased to 10psig, makes that transmembrane pressure is 11psig.Keep transmembrane pressure constant at whole experimental session by regulating the retention valve.Discharge exudate, open second pump and in the charging reservoir, pump into sweet milk, keep constant volume in the charging reservoir with the speed identical with removing exudate.Write down all parameters every 5-10 minute, the speed of regulating second pump is flat constant with whey in the maintenance charging reservoir.Move this test and be concentrated 5.37X or 82% until breast.

Film cleans

The water flux of film recovers higher during using rigorous cleaning program with the assurance cycling.The standardization film cleans and designs cleaning step in the simulation dairy industry, and considers to give birth to the each side of pharmacy standard.In table 1 and 2, provide and carry out following clean cycle behind each treatment step:

Table 5.

The ceramic membrane cleaning

	Step	Concentration	Volume	Time	Temperature	pH
							1) 2) 4) 5) 6) 7) 8) ＞9.5 9) 10)	Water flushing NaOH cleaning clorox NaOH cleaning javelwater washes citric acid cleaning water flushing clorox NaOH water flushing NaOH and preserves	- 0.5M 400ppm 0.5M 400ppm - 0.4M - 300ppm 0.05M - 0.1M	16-20L 1 1 20-25 1 16 1 12 1	5 minutes 10 minutes 30 minutes 5 minutes 30 minutes 10 minutes 15 minutes 10 minutes	60℃ 60 60 60 60 60 60 60 20	7.0 ＞11.5 ＞11.5 7.0 ＜2.75 7.0 7.0 10-12

After having carried out a large amount of engineering operations on the equipment of the clarification breast that in pilot plant, uses, determined that the modification of device therefor and action need is stably to make clean clarification breast.Equipment moved on to the exploitation laboratory from the GMP environment of pilot plant test widely.The change of this system comprised reducing oozes out the position of valve in property management and the change system, be beneficial to clean and sterilisation step during easier rinsing.Revise cleaning program a little to improve cleaning efficiency and to reduce water consumption.Determine the technological temperature scope.Definition process parameter better in the GMP file at last.

The original design of pilot plant is used the rustless steel structure fully.Trouble is cleaned in this design, because a lot of long tubes need be disassembled into cleaning model from technology pattern.Because UF oozes out the length and the internal diameter of property management, it can not be cleaned or rinsing effectively in cleaning program.Many segments are added in the MF system promoting clean, but their structure causes dead space and accumulates chip.These problems are replaced by 1/4 by will long UF the oozing out property management " pipe of internal diameter and being solved.Change cleaning program, make the top of MF film be used to clean the exudate side of film, also just do not needed other segments.During cleaning, the UF exudate guaranteed then and stay on the UF.Simultaneously, in the MF of system part a big heat exchanger is installed also, this carries out accurate temperature control with regard to allowing to MF, but stoped the UF temperature is controlled at the technology applicable scope.From system, shift out heat exchanger, regulate cooling settings so that two systems are all suitably cooled off in suitable temperature range.Be final design below.Assembly equipment is used for preserving, sterilizes and handles.The structure of equipment is provided in figure below O and P in a preferred embodiment of the present invention.

Clean and the sterilization variation

The equipment variations of making need change cleans and the sterilization scheme.After as above showing each run, move cleaning program.Retention valve on the MF need be semi-open, and with auxiliary suitable rinsing during each rinse step, this is because long dead foot is arranged between valve and container.After the 4th operation, the operation cleaning program, and follow the trail of water consumption (minute book 10586).At the 5th, 6 and 7 operation back checking used water in this experiment, and recommend to be used for GMP technology.Just as discussed previously, equipment variations also allows system to sterilize in technology pattern.This is through test.Also determined the required USP water of rinsing disinfectant from system.

Operation

The actual step of carrying out newborn processed with HPTFF is described in lower part.These comprise from the decontamination system to the processed, to cleaning and to whole technical processs of preserving.At the 5-7 time run duration, use these operations on the equipment in the exploitation laboratory, and produce clean clarification breast.

Sterilization

For carrying out HPTFF, use ceramic 0.2 μ m micro-filtration membrane and the 30kda ultrafilter membrane is clarified and concentrated transgenic goat milk, must come decontamination system with the 0.1M sodium hydroxide.As above assembly equipment is to be used for sterilization and PROCESS FOR TREATMENT.The 2L 0.1M sodium hydroxide that pumps into the preparation of USP water passes each system, and cross-flow is 15LPM on MF, and cross-flow is 1LPM on UF.MF is not applied retention pressure, and the retention of UF is applied the pressure of 5psi.Open the exudate valve fully to allow sodium hydroxide around whole systems recirculation.Recirculation was carried out 15 minutes, from system, discharge this solution by the outflow valve between jar and the pump then.Come with USP water rinse system by be full of jar fully with USP water in case of necessity.Flow out discharge 1L water the valve from each.Retention valve on the semi-closed MF, and the exudate valve pointed to garbage fully.Retention on the UF and exudate valve point to garbage fully.Pass through the MF retention with 12L USP water with cross-flow flow velocity 20LPM cleaning down.With 4L USP water with the TMP cleaning down of cross-flow flow velocity 15-20LPM and 6-8psi by the MF exudate.Pass through UF retention and exudate line with 7L USP water with cross-flow flow velocity 1LPM cleaning down, and then wash exudate with 3L USP water.

Use USP water (also can add more if desired) pump MF with 20LPM, increase retention pressure and reach 15psi and do not have exudate pressure, regulate the cross-flow flow velocity to 15LPM with the speed that pumps into then until TMP.Record temperature (must between 25-28 ℃), pressure and cross-flow flow velocity.Measure exudate flow velocity by the exudate dump valve.With 1LPM cross-flow and 5psig retention pressure with there is not exudate pressure (TMP is about 10psig) on UF, to repeat said process.Exudate flow velocity when comparing exudate flow velocity and new film water penetration.If exudate speed less than 80% of initial value, need be cleaned film again or be changed them.

The breast processed

Must merge breast, and be warming up to 15-20 ℃.In the MF container, merge breast, close MF exudate valve then, open the retention valve, and to open pump to cross-flow flow velocity be 20LPM.After 5 minutes, get initial newborn sample.Then pressure being risen to TMP is 15psig, and the cross-flow flow velocity is 15LPM.Continue recirculation and reach 20 ℃ until newborn temperature.Open cooler at 10 ℃ then, open MF exudate valve, the exudate by collecting ceramic membrane is to be concentrated into breast half of original volume in microfiltration systems.Under the transmembrane pressure of 15psi, move MF at 15LPM cross-flow flow velocity.The temperature of MF should increase and maintain 26 ℃ ± 2.0.Must open on the ultrafiltration system cross-flow flow velocity then to 0.8-1LPM/sqft.Measure exudate flow velocity by each film of exudate valve.The retention of the necessary UF of adjusting and exudate pressure are so that the exudate flow velocity mates the exudate flow velocity of MF.In case the exudate flow velocity of UF exudate flow velocity coupling MF should be with 5-6 diafiltration volume of this system's pairing operation.

In case finish diafiltration, parting system is closed MF and discharge and cleaning, and the volume of discharging UF exudate whole clarification concentrate in the charging reservoir at UF is concentrated into half of its volume, and is always concentrated to realize 4X.Discharge UF then, the clarification concentrate of aseptic filtration integral body, and clean UF.

Clean and the preservation scheme

For the parts that suitably clean and preserve this device to allow classification proteins of interest matter, at first from incoming flow input parting system.Semi-open retention valve with soft water (45-65 ℃) the rinsing MF of 20L heat, is discharged exudate.Valve pointed to make the solution recirculation, with the hot 0.5M sodium hydroxide of the 2L recirculation that contains the 400ppm sodium hypochlorite 5 minutes back in the material reservoir.Discharge solution from system, the chemicals identical with 2L replaces.With fresh solution recirculation 30 minutes, from flow out valve, discharge then.Soft water with 20L heat passes through semi-open retention valve rinse-system.By under the TMP of 6-8psi, carrying out the recirculation of water in the retentate side of film with 20lpm, with the soft water of 4L heat only cleaning down pass through exudate.Discharge remaining water by flowing out valve.Under the TMP of 6-8psi, use the 0.5M citric acid recirculation of 2L heat to pass through whole system 30 minutes, discharge citric acid by flowing out valve then with 20LPM.After the caustic alkali step, with the retentate side of 15L soft water cleaning down MF, with 4L soft water cleaning down exudate side.Passed through MF15 minute with the hot 0.05M sodium hydroxide of the 2L recirculation that contains the 400ppm bleach then, discharge then, use 10L water cleaning down retentate side later on, with 4L water cleaning down exudate in the caustic alkali step.

Discharge and, UF retention and the sensing of exudate line are discharged by the retention valve is pointed to, to carry out preliminary water flushing whole exudate line sensing discharge (not being to pass through valve).Always with the 1LPM process pump, if promptly retention pressure increases, pump speed also must increase to keep 1LPM.With these two lines of 4LUSP water rinse.Use the 2L 0.5M sodium hydroxide of making by USP water that contains the 250ppm sodium hypochlorite to wash this two lines.With the whole system that 2L fresh solution recirculation links to each other with the charging reservoir by the exudate line, the opening of retention valve passed to reservoir 60 minutes.Discharge solution by flowing out valve.As initial cleaning these two lines are pointed to discharge.With USP water filling containers, and by flowing out valve discharge 1L.Wash this two lines with 8L water, then reuse 4L water flushing exudate line under 5psi retention pressure.Passed through whole system 60 minutes with 2L 0.4M citric acid recirculation then.Discharge acid solution by flowing out valve, fill reservoir with USP water then, and discharge 1L by flowing out valve.By retention and exudate line, stride under the film cross-flow flow velocity reuse 8L flushing passing through exudate then at 5psi retention pressure and 1LPM with the flushing of 8L water.

After cleaning and intact these two systems of rinsing, they are assembled up preservation (last figure).In each feed containers, pour 2L 0.1M sodium hydroxide into, and open retention and the exudate valve pumps into by the whole system recirculation, close to garbage, continue 2 minutes.Covered container then, flag state are clean, and are kept in the 0.1M sodium hydroxide.

Show that technological parameter is important for the material of conformity of production.Being used for clarifying film is CerCor pottery 0.2 μ m aperture film, 1.5sqft and 30kDa NMWCO Pall Filtron PES box, 2sq.ft. (2 boxes).For the clarification and the flux of the best of proteins of interest matter, the temperature of microfiltration systems should maintain 26-29 ℃.Microfiltration systems should operation under the transmembrane pressure of the retention flow velocity of 14 LPM (42cm/s) and 15psig.Breast should be concentrated to the 40-70% (1.5-2.5X) of former merging volume.The ultrafiltration part of system should operate under 1.6-2LPM retention flow velocity and the 20-30psig feed pressure.By regulating exudate pressure, the exudate flow velocity should mate with the exudate flow velocity of microfiltration systems.Final integral body clarification concentrate should be 1/4th (4X concentrates) that merge former newborn volume.

The recombinant preparation

In treatment and diagnostic application, the ever-increasing a large amount of recombiant proteins of quantity have been developed.But having many in these protein is difficulty or expensive with conventional method with functional form and/or requirement preparation.Conventional method comprises to host cell for example inserts the gene of being responsible for producing specified protein in antibacterial, yeast or mammalian cell such as COS or the Chinese hamster ovary celI, allows cell grow in culture medium then.Cultured cells is synthesized expectation protein then.Traditional antibacterial or Yeast system may not prepare a lot of complex proteins of functional form.Although mammalian cell can the duplication of production complex proteins, their growth generally is a difficulty and expensive, and often can only produce the protein of mg/L amount.In addition, the non-secretory protein of purification is difficult relatively from protokaryon or mammalian cell, because they are not secreted in the culture medium.

Usually, the transgenic technology feature is that manufacturing and secretion be not usually by the method for excretory protein (non-secretory protein).This method comprises marking protein from nucleic acid construct, and described nucleic acid construct comprises:

(a) promoter, for example breast epithelium specificity promoter, for example lactoprotein promoter;

(b) signal sequence, it can instruct proteinic secretion, for example the signal sequence of newborn specific protein;

(c) randomly, the coding secretory protein is gone into for example Ruzhong of transgene mammal as the sequence of enough parts of the aminoterminal coding region that is secreted into protein of milk to allow non-secretion protein excretion; With

(d) the proteic sequence of the non-secretion of coding,

Wherein the element (a) and (b), randomly (c) preferably be connected with (d) according to described order is exercisable.

In preferred embodiments: element a, b, c (if existence) and d are from homologous genes; Element a, b, c (if existence) and d are from two or more genes;

In preferred embodiments, secretion is the Ruzhong that is secreted into transgene mammal.

In preferred embodiments, signal sequence is the beta-casein signal sequence; Promoter is the beta-casein promoter sequence.

In preferred embodiments, non-sequence encoding secreted protein is: derive from the people; The coding truncate, nuclear or cytoplasm polypeptide; The proteins of interest matter of coding human serum albumin or other expectations.

Except as otherwise noted, implement the present invention and be to use cytobiology, cell culture, molecular biology, genetically modified organism, microbiology, recombinant DNA and immunologic routine techniques, this is in those skilled in the art's limit of power.These technical descriptions are in document.For example referring to Molecular Cloning A Laboratory Manual, 2nd Ed., ed.By Sambrook, Fritsch and Maniatis (Cold Spring Harbor LaboratoryPress:1989); DNA Cloning, Volumes I and II (D.N.Glover ed., 1985); Oligonucleotide Synthesis (M.J.Gait ed., 1984); Mullis et al.U.S.Patent No:4,683,195; Nucleic Acid Hybridization (B.D.Hames ﹠amp; S.J.Higgins eds.1984); Transcription And Translation (B.D.Hames ﹠amp; S.J.Higgins eds.1984); Culture OfAnimal Cells (R.I.Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); The treatise, and Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J.H.Miller and M.P.Calos eds., 1987, Cold SpringHarbor Laboratory); Methods In Enzymology, Vols.154 and 155 (Wu et al.eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, VolumesI-IV (D.M.Weir and C.C.Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

The breast specificity promoter

Be used for implementing transcripting promoter of the present invention preferably in the activated promoter of mammal epithelial cell, comprise control coding lactoprotein such as casein, beta lactoglobulin (Clark et al., (1989) whey acid protein (Gorton et a. BIO/TECHNOLOGY 7:487-492),, Bio/Technology 5:1183-1187) and the promoter of the gene of lactalbumin (Soulier et a., (1992) FEBS LETTS.297:13) (1987).Casein promoter can derive from α, β, γ or the κ casein gene of any mammalian species; Preferred promoter is from goat β casein gene (DiTullio, (1992) Bio/Technology 10:74-77).Breast specific protein promoter or in mammary gland tissue, can be derived from cDNA or genome sequence by the activated promoter of specificity.Preferably they are from genome.

For above all mammary gland-specific genes of enumerating, DNA sequence information at least a often be to be available in several animals.For example, referring to Richards et al., J.Biol.Chem.256,526-532 (1981) (alpha-lactalbumin rat); Campbell et al., Nucleic Acids Res.12,8685-8697 (1984) (rat WAP); Jones et al., J.Biol.Chem.260,7042-7050 (1985) (rat beta-casein); Yu-Lee ﹠amp; Rosen, J.Biol.Chem.258,10794-10804 (1983) (rat gamma-casein); Hall, Biochem.J.242,735-742 (1987) (alpha-lactalbumin people); Stewart, Nucleic Acids Res.12,389 (1984) (cattle α s1 and κ casein cDNAs); Gorodetsky et al., Gene 66,87-96 (1988) (cattle beta-casein); Alexander et al., Eur.J.Biochem.178,395-401 (1988) (cattle κ casein); Brignon et al., FEBS Lett.188,48-55 (1977) (cattle α S2 casein); Jamieson et al., Gene 61,85-90 (1987), Ivanov et al., Biol.Chem.Hoppe-Seyler 369,425-429 (1988), Alexander et al., Nucleic Acids Res.17,6739 (1989) (cattle beta lactoglobulins); Vilotte et al., Biochimie 69,609-620 (1987) (cattle alpha-lactalbumin).The 26S Proteasome Structure and Function of different milk protein genes is summarized the ﹠amp in Mercier; Vilotte, J.Dairy Sci.76,3079-3098 (1993) (its integral body being incorporated herein by reference) for all purposes.For the scope of other sequence datas that may need, can use existing sequence easily to clone the sequence that has obtained regional side as probe.By using known nucleotide sequence of the same clan or proteinic antibody of the same clan, also can obtain to regulate sequence from these biological mammary gland-specifics as the libraries of probe screening from different biologies.

Although for the purpose understood by explanation and embodiment some at length invention has been described, it is implemented some variations and modification will be conspicuous for those skilled in the art.Therefore, these descriptions and embodiment should not be construed as and limit the scope of the invention, and scope of the present invention is limited to the appended claims.

Therefore, be to be understood that to the invention provides the embodiment of improving one's methods that is used for high performance tangential flow filtration that obtaining the molecules of interest of high yield from given incoming flow, but these embodiments only are the examples to the application of principle of the present invention.Apparently, from above stated specification, change form, the application of using method and disclosed element can not depart from the scope of spirit of the present invention or claims.

Prior art incorporated herein by reference

1.Aranha-Creado H，and Fennington GJ Jr(1997，Cumulative Viral TiterReduction Demonstrated By Sequential Challenge Of A Tangential FlowMembrane Filtration System And A Direct Flow Pleated Filter Cartridge，PDAJPHARM SC TECHNOL 51(5)：208-212.

2.Aravindan GR，et al.，(1997)，Identification，Isolation，And Characterization OfA41-Kilodalton Protein From Rat Germ Cell-Conditioned Medium ExhibitingConcentration-Dependent Dual Biological Activities，ENDOCRINOLOGY138(8)：3259-68.

3.Christy C.，et al.，High Performance Tangential Flow Filtration：A HighlySelective Membrane Separation Process，DESALJNATION，vol.144：133-36(2002).

4.Gabler et al.，(1987)，Principles of Tangential Flow Filtration：Applications toBiological Processing，in FILTRATION IN THE PHARMACEUTICAL INDUSTRY，pp.453-490.

5.Ghosh R，et al.，Parameter Scanning Ultrafiltration：Rapid Optimisation ofProtein Separation，BIOTECHNOLBOOENG.，2003 Mar 20；81(6)：673-82.

6.Kahn DW，et al.，(2000)，Purification of Plasmid DNA by Tangential FlowFiltratiion，BIOTECHNOL BIOENG.69(1)：101-106.

7.Kawahara H，et al.，(1994)，High-Density Culture of FM-3A Cells Using aBioreactor With An External Tangential-Flow Filtration Device，CYTOTECHNOLOGY 14(1)：61-66.

8.Koros，WJ.et al.，(1996)，Terminology for Membranes and MembraneProcesses(IUPAC Recommendations 1996).PURE & APPL.CHEM.68：1479-89.

9.Millesime L，ct al.，Fractionation of Proteins with Modified Membranes，BIOSEPARATION，1996 Jun；6(3)：135-45，

10.Prado SM，et al.，(1999)，Development And Validation Study For TheChromatographic Purification Process For Tetanus Anatoxin On Sephacryl S-200 High Resolution，BOLL CHIM FARM.138(7)：364-368.

11.Porter，ed.，HANDBOOK OF INDUSTRIAL MEMBRANE TECHNOLOGY，(NoyesPublications，Park Ridge，New Jersey，1998)Pp.160-176.

12.Ramachandra-Rao，H.G.et al.，Mechanisms of Flux Decline DuringUltrafiltration of Dairy Products and Infiuence of pH on Flux Rates of Wheyand Buttermilk，DESALINATION，vol.144：319-24(2002).

13.Strauss PR(1995)，Use Of Filtron Mini-Ultrasettetm Tangential Flow DeviceAnd Filtron Microseptm Centrifugal Concentrators In The Early Stages OfPurification Of DNA Polymerases，BIOTECHNIQUES 18(1)：158-160.

14.Van Reis R.，and Zydney A.，Review，CURR OPIN BIOTECHNOL.，2001 Apr；12(2)：208-11.

15.Van Reis R.，et al.，high Performance Tangential Flow Filtration，BIOTECH.BIOENG.，56：71-82，(1997).

16.Zeman，L.J.& Zydney，A.L.(1996)；Microfiltration and Ultrafiltration，inPRINCIPLES AND APPLICATIONS.(Marcel Dekker ed.)，New York.

United States Patent (USP) of incorporating into as a reference and patent application

1.Van Reis R.M.，et al.，United States Patent No.：6,555,006，TANGENTIAL-FLOW FILTRATION SYSTEM.

2.Van Reis R.M.，et al.，United States Patent No.：6,387,270，TANGENTIAL-FLOW FILTRATION SYSTEM.

3.Van Reis R.M.，et al.，United States Patent No.：6,221,249，TANGENTIAL-FLOW FILTRATION SYSTEM.

4.Van Reis R.M.，et al.，United States Patent No.：6,054,051，TANGENTIAL-FLOW FILTRATION SYSTEM.

5.Van Reis R.M.，et al.，United States Patent No.：5,490,937，TANGENTIALFLOW FILTRATION PROCESS AND APPARATUS.

6.Van Reis R.M.，et al.，United States Patent No.：5,256,294，TANGENTIALFLOW FILTRATION PROCESS AND APPARATUS.

7.Sandblom R.M.et al.，United States Patent No.：4,105,547，FILTERINGPROCESS.

8.Jain M.et al.，United States Patent No.：4,351,710，FRACTIONATION OFPROTEIN MIXTURES.

9.Van Reis R.M.，el al.，United States Patent Application No.：20020108907；TANGENTAL-FLOW FILTRATION SYSTEM，filed 4/12/2002.

Claims (78)

1. method of separating proteins of interest matter from incoming flow comprises:

(a) filter described incoming flow by high performance tangential flow filtration technology, this technology is separated described molecules of interest based on the aperture of described proteins of interest matter with electric charge from described incoming flow, the level that keeps flux about 5 to 100% scopes of transition point flux in flux is distinguished the pressure dependence of TMP curve simultaneously, wherein the direction along film keeps transmembrane pressure to be substantially constant at the level of the transmembrane pressure that is not more than filtering transition point, thereby described proteins of interest matter is separated by selectivity from described incoming flow, makes described proteins of interest quality guarantee hold its biological activity;

(b) filter described incoming flow by ultrafiltration technology; With

Wherein said filtration occurs in more than the described proteins of interest fundamental change point flux;

Wherein said molecule has the molecular weight between 1 to 1000kDa.

2. the method for claim 1 further comprises the described incoming flow of classification.

3. the method for claim 1 further comprises the described incoming flow of clarification.

4. the method for claim 1 further comprises the described incoming flow of diafiltration.

5. the method for claim 1, further being included in filtering the first half increases transmembrane pressure and reduces flow.

6. the method for claim 5, further be included in this technology the second half in reduce transmembrane pressure.

7. the process of claim 1 wherein that described incoming flow is concentrated before filtration.

8. the process of claim 1 wherein in the described incoming flow of molecular weight ratio of described protein of interest matter second kind of proteins of interest matter greatly less than 10 times, perhaps little of 10 times than it.

9. the process of claim 1 wherein that second kind of proteins of interest matter surpasses 10 times greatly in the molecular weight ratio mixture of described proteins of interest matter, perhaps surpass 10 times for a short time, but they have identical electric charge or isoelectric point, IP than it.

10. the method for claim 1 further comprises concentrating described incoming flow.

11. the process of claim 1 wherein that all filtration stages are ultrafiltration.

12. the process of claim 1 wherein that described incoming flow is a breast.

13. the process of claim 1 wherein that described incoming flow is a lysis solution.

14. the process of claim 1 wherein that described protein is bio-pharmaceutical.

15. the method for claim 12, the condition of wherein said breast is selected from following state:

A) lactogenesis;

B) dilute;

C) handle with buffer solution;

D) chemically treated; With

E) part is evaporated.

16. the method for claim 2, wherein said classification step is utilized ceramic filtration membrane.

17. the method for claim 3, wherein said clarification steps is utilized ceramic filtration membrane.

18. the method for claim 2, wherein said classification step utilization have the polymer filter membrane of electrical characteristics such as definite.

19. the method for claim 3, wherein said clarification steps is utilized the polymer filter membrane.

20. the method for claim 2, wherein said classification step is utilized cellulose filter membrane.

21. the method for claim 3, wherein said clarification steps is utilized cellulose filter membrane.

22. the method for claim 2 further comprises the optimization system parameter.

23. the method for claim 22, wherein said systematic parameter comprise temperature, feed stream flow rate, transmembrane pressure, incoming flow concentration and diafiltration volume.

24. the method for claim 3 further comprises the optimization system parameter.

25. the method for claim 24, wherein said systematic parameter comprise temperature, feed stream flow rate, transmembrane pressure, incoming flow concentration and diafiltration volume.

26. the process of claim 1 wherein that described molecules of interest is a biological entities, is selected from protein, immunoglobulin, polypeptide, peptide, glycoprotein, RNA and DNA.

27. the method for claim 23, wherein Zui Jia temperature range is 15 ℃ to 50 ℃.

28. the method for claim 23, wherein Zui Jia temperature range is 20 ℃ to 35 ℃.

29. the method for claim 23, wherein Zui Jia temperature range is 25 ℃ to 29 ℃.

30. the method for claim 25, wherein Zui Jia temperature range is 15 ℃ to 50 ℃.

31. the method for claim 25, wherein Zui Jia temperature range is 20 ℃ to 35 ℃.

32. the method for claim 25, wherein Zui Jia temperature range is 25 ℃ to 29 ℃.

33. the method for claim 23, wherein feed stream flow rate is 10cm/ second to 100cm/ second.

34. the method for claim 23, wherein feed stream flow rate is 20cm/ second to 60cm/ second.

35. the method for claim 23, wherein feed stream flow rate is 25cm/ second to 45cm/ second.

36. the method for claim 25, wherein feed stream flow rate is 10cm/ second to 100cm/ second.

37. the method for claim 25, wherein feed stream flow rate is 20cm/ second to 60cm/ second.

38. the method for claim 25, wherein feed stream flow rate is 25cm/ second to 45cm/ second.

39. the method for claim 23, wherein the scope of transmembrane pressure is that 2psi is to 40psi.

40. the method for claim 23, wherein the scope of transmembrane pressure is that 5psi is to 30psi.

41. the method for claim 23, wherein the scope of transmembrane pressure is that 10psi is to 20psi.

42. the method for claim 25, wherein the scope of transmembrane pressure is that 2psi is to 40psi.

43. the method for claim 25, wherein the scope of transmembrane pressure is that 5psi is to 30psi.

44. the method for claim 25, wherein the scope of transmembrane pressure is that 10psi is to 20psi.

45. the method for claim 23, wherein incoming flow concentration are that the 0.25X of genuine milk is to 4X.

46. the method for claim 23, wherein incoming flow concentration are that the 0.5X of genuine milk is to 3X.

47. the method for claim 23, wherein incoming flow concentration are that the 1.0X of genuine milk is to 2X.

48. the method for claim 25, wherein incoming flow concentration are that the 0.25X of genuine milk is to 4X.

49. the method for claim 25, wherein incoming flow concentration are that the 0.5X of genuine milk is to 3X.

50. the method for claim 25, wherein incoming flow concentration are that the 1.0X of genuine milk is to 2X.

51. the method for claim 23, wherein the diafiltration volume range is that the 1X of spissated MF retention volume is to 20X.

52. the method for claim 23, wherein the diafiltration volume range is that the 3X of spissated MF retention volume is to 15X.

53. the method for claim 23, wherein the diafiltration volume range is that the 5X of spissated MF retention volume is to 10X.

54. the method for claim 25, wherein the diafiltration volume range is that the 1X of spissated MF retention volume is to 20X.

55. the method for claim 25, wherein the diafiltration volume range is that the 3X of spissated MF retention volume is to 15X.

56. the method for claim 25, wherein the diafiltration volume range is that the 5X of spissated MF retention volume is to 10X.

57. the method for claim 2, wherein ultrafilter membrane is used for all filtration steps.

58. the method for claim 7, wherein ultrafilter membrane is used for all filtration steps.

59. the method for claim 12, wherein said breast is with being selected from following solution-treated:

A) water;

B) aqueous solution of buffered salt;

C) intercalating agent;

D) acid solution; With

E) aqueous slkali.

60. the method for claim 4, wherein said diafiltration utilizes the ultrafiltration exudate.

61. the method for claim 4, wherein said diafiltration utilizes water.

62. the method for claim 4, wherein said diafiltration utilizes buffered saline solution.

63. the process of claim 1 wherein that the film that uses surpasses 20 ℃ solution cleaning with temperature.

64. the process of claim 1 wherein that the film temperature of using is the solution cleaning of 20 ℃ of-70 ℃ of scopes.

65. the process of claim 1 wherein that the film temperature of using is the solution cleaning of 40 ℃ of-60 ℃ of scopes.

66. the process of claim 1 wherein that the film that uses cleans with acid solution.

67. the process of claim 1 wherein that the film that uses cleans with aqueous slkali.

68. the process of claim 1 wherein that the film that uses cleans with hypochlorite solutions.

69. claim 66,67 or 68 method further are included in and use water flushing after the selected solution.

70. the process of claim 1 wherein before use with the hydroxide solution employed film of sterilizing.

71. the process of claim 1 wherein before use with the alcoholic solution employed film of sterilizing.

72. the process of claim 1 wherein before use with the hypochlorite solutions employed film of sterilizing.

73. the process of claim 1 wherein that employed film cleans 20 minutes to 45 minutes clock times.

74. the method for claim 1, further be included in the second tangential flow filtration stage by than the littler membrane filtration of the first filtration stage membrane aperture from the filtering thing that leaches, and the thing recirculation that leaches of this second filtration stage returned first filtration stage, thereby repeat this technology.

75. a method of separating proteins of interest matter from incoming flow comprises:

(a) filter described incoming flow by high performance tangential flow filtration technology, this technology is separated described molecules of interest based on the aperture of described proteins of interest matter with electric charge from described incoming flow, the level that keeps flux about 5 to 100% scopes of transition point flow in flux is distinguished the pressure dependence of TMP curve simultaneously, wherein the direction along film keeps transmembrane pressure to be substantially constant at the level of the transmembrane pressure that is not more than filtering transition point, described whereby proteins of interest matter is separated by selectivity from described incoming flow, makes described proteins of interest quality guarantee hold its biological activity;

(b) filter described incoming flow by ultrafiltration technology; With

(c) in filtering the first half, increase transmembrane pressure and reduce flow;

(d) reduce with filtration and increase subsequently or keep flux,

Wherein said filtration occurs in more than the described proteins of interest fundamental change point flux;

Wherein said molecule has the molecular weight between 1 to 1000kDa.

76. the method for claim 1 or 75, wherein said proteins of interest matter is recombinant human alpha-fetoprotein.

77. the method for claim 1 or 75, wherein said protein of interest matter is the recombined human albumin.

78. the method for claim 1 or 75, wherein said proteins of interest matter derives from the breast of transgene mammal.

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