CN1569031A - Plasma component series preparation by inactivating virus and sieving sectionally by hyperfiltration membrane - Google Patents
Plasma component series preparation by inactivating virus and sieving sectionally by hyperfiltration membrane Download PDFInfo
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- CN1569031A CN1569031A CNA2004100130849A CN200410013084A CN1569031A CN 1569031 A CN1569031 A CN 1569031A CN A2004100130849 A CNA2004100130849 A CN A2004100130849A CN 200410013084 A CN200410013084 A CN 200410013084A CN 1569031 A CN1569031 A CN 1569031A
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Abstract
The invention provides a plasma component series preparation by inactivating virus and sieving sectionally by hyperfiltration membrane, wherein the preparation comprises, filtering the raw material blood plasma through clarification or nano film virus removing, or charging protection agent for virus hot eradication, sieving with ultrafiltration film to obtain series components, filtering through ultrafiltration dialysis and ultrafiltration concentration and degerming.
Description
Technical field the present invention relates to the plasma component preparation series of the inactivation of virus that a kind of related human disorder prevents and treats usefulness/ultrafilter membrane segmentation sieve method preparation.
Background technology blood plasma is the yellowish glue liquid solution that whole blood is removed the acellular composition that obtains behind (separation) hemocyte.Blood plasma can be divided into single part of blood plasma and the big pooled plasma of many person-portions.Single part of blood plasma is to separate the blood plasma that obtains from single blood donor's blood, is to belong to one of blood constituent goods, is used for component blood transfusion.Big pooled plasma is numerous blood donors' a blood plasma, is the raw material of plasma component preparation, is used to prepare the plasma component preparation.
With the plasma protein products of prior art for preparing, often be called blood products, or blood plasma product, blood plasma preparation, blood plasma derivant.Plasma protein products is by healthy people's blood plasma, immune human plasma, immune animal blood plasma or serum, or be the biological product quasi drugs that is used to prevent and treat human diseases related of feedstock production with the protein that biotechnology produces, as the thrombin of human albumin, human normal immunoglobulin (IgG), human blood or DNA reorganization and each type of special immunoglobulin etc.
The preparation method of tradition plasma protein products has the cold ethanol fractional precipitation, salting out method, chromatography, PEG method etc., what all adopt is chemical method, it is difference according to the physicochemical property of range protein in the blood plasma, adding chemical reagent reacts with it, the range proteins different to physicochemical property carry out fractional precipitation/dissolving, or separation such as absorption/eluting, collection method, obtain to contain a certain protein component respectively, and also contain a large amount of impurity simultaneously, as chemical reagent such as ethanol, protein denaturation, the mixed solution system of compositions such as the pyrolysis product and the product that is activated.They must go the product of dialysis, refining, the refining purge process gained that a series of removal impurity components such as concentrates again, just can reach the conventional blood products prescription of prior art.The tradition plasma protein products, or claim traditional plasma protein fraction preparation, only contain single protein component.
The subject matter that plasma protein products of prior art for preparing and preparation method thereof exists:
1, plasma component has about 200 kinds altogether of protein and nonprotein components, and this method can only be made albumin, immunoglobulin product, and a spot of thrombin and some trace albumin goods etc., and other protein and nonprotein component have all gone out of use;
2, be that 80-90 gram meter, total protein are 55 gram meters according to every liter of blood plasma gross weight 1000 gram meters, solubility total solid, can only extract wherein about 33 grams per liter blood plasma of albumen by this method, and wherein 96.7% of the blood plasma gross weight, the blood plasma total solid 60%, the blood plasma total protein 40%, all gone out of use;
3, in preparation process, various chemical reagent such as essential adding ethanol, and concentration of alcohol, salt ionic concentration, hydrogen ion concentration (pH value), protein concentration, temperature five becomes the modulation of factor, make plasma proteins through condensing repeatedly, precipitate, dissolving, also will add filter aid, and centrifugal filtration or filter pressing repeatedly, like this, a large amount of impurity can occur inevitably, reach damaging action, and influence quality of item plasma proteins;
4, adding the protein precipitation granule of ethanol formation and the filter aid of adding etc. in the preparation, all is electronegative activator, and FXIII is had extremely strong activation, finally produces PKA, can cause bad clinical response;
The essential ethanol that adds when 5, preparing, make the dehydration of plasma protein molecule, cohesion, precipitation, deposit seed is exactly the polymer of protein molecule, not the protein polymer (polymer) of thorough depolymerization, anticomplementary activity effect (ACA) is arranged, can cause bad clinical response;
6, have only highly purified albumin (purity 96%) and high-purity immunoglobulin (IgG) (purity 95%), just can carry out intravenous injection.
Existing " a kind of process for deactivating freeze dried human plasma virus " (number of patent application 02103960), it includes following steps: refrigerated plasma is placed melt the slurry jar, melt slurry, melt slurry and place the inactivation of viruses jar, add phosphoric acid tributyl and TritonX, stir, heat up, move in the cup type centrifuge, add vegetable oil extraction inactivator, carry out centrifugalize, water blood plasma after separating is made the not blood plasma of phosphoric acid tributyl and TritonX-100 through containing C18 resin chromatographic column, be to carry out packing after 0.2 micron the membrane filtration degerming with blood plasma via hole diameter after ultrafilter membrane concentrates, the blood plasma of packing placed carry out the vacuum and low temperature distillation in the freeze dryer, dry sealing back is carried out xeothermic deactivation and is made powdery finished product blood plasma in boiling water bath.Its blood plasma product inactivation of virus is more thorough, uses saferly, and has storage and transport advantage more easily.
Summary of the invention the objective of the invention is at the existing problem of the blood plasma product of prior art for preparing, aim to provide a kind ofly do not have any virus activity, do not have any microbial activity, no communicate illness danger be perfectly safe, performance is better, function is more complete, curative effect is better, steady quality, plasma component preparation series that all can intravenous inactivation of virus/ultrafilter membrane segmentation sieve method preparation.
The implementation of the object of the invention is, the plasma component preparation series of inactivation of virus/ultrafilter membrane segmentation sieve method preparation, and its process is as follows:
Raw blood plasma, clarification filtration and nanometer film are removed virus filtration step by step with microporous membrane, filter through the screening of 60-10KD ultrafilter membrane, what obtain surpasses filtrate, or repeat aforesaid again except that virus filtration and ultrafilter membrane screening filtration, and the filtrate of ultra-filtration repeatedly that obtains through aseptic filtration, is the small molecule component preparation; The ultrafiltration trapped fluid that obtains, or use raw blood plasma, add exogenous protective agent and carry out hot deactivation, remove virus filtration through clarification filtration and nanometer film, obtain the deactivation material.Deactivation material (without ultrafiltration membrance filter) is whole plasm component or composite class component; The deactivation material, the ultrafilter membrane segmentation screening through different apertures separates, and obtains serial component; Whole plasm component and serial component through ultrafiltration dialysis and ultrafiltration and concentration, obtain each serial component that conformance with standard requires, and again through aseptic filtration, are the plasma component preparation series.
Exogenous protective agent includes sodium caprylate, sodium caprylate/acetyl-l-tryptophan sodium, sorbitol, mannitol, xylitol, maltose, glucose, sucrose; its dosage is: each 0.06～0.10mM/ gram albumen of sodium caprylate 0.12-0.20mM/ gram albumen or sodium caprylate/acetyl-l-tryptophan sodium; sorbitol or mannitol, xylitol 20～120 grams per liter blood plasma, and/or maltose and/or glucose, sucrose 30～150 grams per liter blood plasma.
The present invention has following advantage:
1) can be with about 200 kinds of protein and the nonprotein component in the blood plasma, screening separates prepares corresponding component preparation, makes that blood plasma can obtain fully, reasonable, the comprehensive comprehensive utilization;
2) raw blood plasma can be used ACD anticoagulant anticoagulant blood plasma, has the effect of better protection blood plasma, help and help to preserve, natural sex, stability and the health giving quality of maintainer line series preparation;
What 3) the present invention adopted is the physics thermoinactivation, does not retain any residue after deactivation stops;
4) keep the inherent inherent natural protective agent of raw blood plasma self (albumin), can give full play to self natural protective agent effect of a large amount of albumin (accounting for more than 50% of blood plasma total protein) in the blood plasma, on this basis, add multinomial exogenous protective agent, interior, the common protection of external source duplicate protection agent, coordinating protection, give mutual protection, carry out inactivation treatment under a chain of protective effect, plasma component can stand inactivation of virus/removal processing of the stronger more degree of depth and not condense, solidify, cracking, degeneration, natural sex that can better more effective assurance plasma fraction, physiologically active and health giving quality, can obtain being perfectly safe of no communicate illness danger, performance is better, function is more complete, curative effect is better, does not have the preparation series of any virus activity and no any microbial activity;
5) do not handle through chemical reagent such as ethanol, protein condenses, precipitation, dissolving, activation do not occur, need not to add filter aid, do not need centrifugation or filter pressing, so there is not consequent impurity, the therefore preparation of the making bad clinical response that do not have ACA, PKA etc. to cause;
6) do not change through any phase (solid, liquid), directly separate the preparation that obtains by the membrane filtration technique screening, can be better, more perfect, more effective, protect natural sex, physiological, pure property and the safety of plasma component (protein component and non-protein component) more comprehensively, obtained performance is better, can preparation series product long preservation, that curative effect is better;
7) preparation series is that the ultrafilter membrane that the application membrane aperture varies in size carries out the segmentation screening, to obtain the different plasma component preparation series of molecular weight, preparation method is to belong to the physical filtering sieve method, simple and convenient, fast and reliable, does not have phase (solid phase, liquid phase) to change to cause to the damage of protein performance with to the influence of the quality of the pharmaceutical preparations;
8) can directly prepare serial component preparation, as high molecular weight component preparation, macromolecule component preparation, middle molecular weight component preparation, lower molecular weight components preparation, combination partner preparation, the whole plasm component preparation in human normal plasma and the immune human plasma series component preparation, and the destination protein goods raw material components of immune serum immune protein goods raw material components and biotechnology generation etc., preparation all is intravenous injections, has quick-acting, efficient, lasting curative effect effects;
9) raw material components of preparing, volume is little, is convenient to preserve, and can long preservation, helps further processing, purification to prepare high-quality, preparation that purity is high;
10) preparation series of the present invention, be to obtain with membrane filtration screening isolation technics, as normal blood center and blood-collecting station can the separate blood method of separating component simple and convenient, need not profound hypothermia workshop and relevant complex device, small investment, instant effect, cost is low, efficient is high, with short production cycle, the production that can more massively feed intake, and labor intensity is little, help production environment and improve, help environmental protection;
11) preparation technology meets modernization, scale, the automated production requirement that Blood Preparations is produced, and meets the GMP quality management requirement that blood plasma preparation is produced.
Appended drawings is a process chart of the present invention
The specific embodiment is with reference to accompanying drawing, implementation process of the present invention is, get raw blood plasma, one group of microporous membrane with membrane aperture difference (as 10-5-1-0.45-0.22 μ M) carries out clarification filtration and nanometer film (as BMM35 μ M) removal virus filtration step by step, filter through 60-10KD ultrafilter membrane screening, obtain to surpass filtrate or ultra-filtration filtrate and ultrafiltration trapped fluid repeatedly.Wherein surpass filtrate or ultra-filtration filtrate repeatedly,, be the small molecule component preparation through aseptic filtration; Trapped fluid wherein, or directly use raw blood plasma, after adding exogenous protective agent, at 60 ℃ ± 20 ℃ damp heat inactivatings that carry out more than 5-15 hour, or after being lyophilized into powder, 80 ℃ ± 20 ℃ xeothermic deactivations of carrying out more than 50-100 hour, clarification filtration and nanometer film are removed virus filtration step by step through microporous membrane again, obtain virus-free active material (claiming the deactivation material),, be the whole plasm component without the ultrafilter membrane screener; The deactivation material separates through the different ultrafilter membrane segmentation screening of molecular cut off, obtains each serial component.Whole plasm component and each serial component, reuse dialysis is carried out ultrafiltration dialysis and ultrafiltration and concentration and is carried out aseptic filtration with 0.20-0.22 μ m microporous membrane with ultrafilter membrane, obtain no any microbial activity, apyrogeneity matter, be suitable for the plasma component preparation series that the human vein infusion is used, preparation can be liquid dosage form or freeze-dried formulation.
Raw blood plasma comprises the destination protein solution that produces with ACD anticoagulant anticoagulant human normal plasma, immune human plasma, immune serum or blood plasma, biotechnology.
The various plasma component preparation series that ultrafilter membrane segmentation screening partition method obtains are exemplified below:
One, human normal plasma natural constituents preparation series:
Deactivation material without the ultrafilter membrane screening is whole plasm component preparation
1, the ultrafilter membrane with molecular cut off 160～300KD sieves separation to human normal plasma, immune human plasma deactivation material, obtains high molecular component preparation and large, medium and small molecular combinations component preparation or material, and is specific as follows:
What 1. obtain holds back concentrated solution, claim human normal plasma high molecular component preparation, or claiming thrombin (FI, FVIII) preparation, key component is FI, the FVIII of molecular weight greater than 160～300KD, is mainly used in the blood coagulation disorders due to treatment hemophilia A and the fibrinogenopenia.
2. the ultrafiltrate of Huo Deing, claim human normal plasma large, medium and small molecular combinations component preparation or material, or claiming IgG/ albumin/micromolecule combination partner preparation, key component is IgG, albumin and the micromolecule plasma component (protein component and non-protein component) of molecular weight less than 160～300KD.It can further sieve and be the respective components preparation, also can be used as the combination preparation that contains these three kinds of components.
2, the ultrafilter membrane with molecular cut off 70～150KD sieves separation to large, medium and small molecular combinations component preparation or material, obtain the macromolecular components preparation and in, micromolecule combination partner preparation or material, be specially:
What 1. obtain holds back concentrated solution, claims human normal plasma macromolecular components preparation, or claim immunoglobulin (IgG) preparation that main component is the IgG of molecular weight 156KD.It is mainly used in treatment IgD disease, self property immune disease and infectious disease.
2. the ultrafiltrate of Huo Deing, claim in the human normal plasma, small molecule component preparation or material, or claiming albumin/micromolecule combination partner preparation, key component is albumin and the blood plasma small molecule component (small molecular protein component and non-protein component) of molecular weight less than 70～150KD.It can do further to be separated into the respective components preparation, also can be used as the combination preparation that contains these two kinds of components.
3, ultrafilter membrane centering, micromolecule combination partner preparation or the material with molecular cut off 60-10KD sieves separation, and molecular components preparation and small molecule component preparation in the acquisition specifically have:
What 1. obtain holds back concentrated solution, claims molecular components preparation in the human normal plasma, or claim albumin preparation that main component is the albumin of molecular weight 66KD.It is mainly used in traumatic, the hemorrhagic shock of control, serious burn and hypoproteinemia etc.
2. the ultrafiltrate of Huo Deing, comprise that raw blood plasma is at first after clarification filtration and nanometer film are removed virus filtration step by step, directly sieve and separate the ultrafiltrate that obtains with the 60-10KD ultrafilter membrane, claim human normal plasma small molecule component preparation, or title biological response modifier/water salt poising agent, main component is plasma proteins component and the nonprotein component [saccharide of molecular weight less than 10～60KD, lipid, inorganic salt, aminoacid, other organic acid, vitamin, trace element, hormone, cytokine, moisture], be the natural physiological solution of human body.Mainly make biological response modifier and water salt poising agent, the immune function of human body of enhancing and immunoregulation effect are arranged, and replenish and effects such as adjusting human body water salt balance and electrolyte supplement, can be used for treating chemotherapy of tumors, radiotherapy and by low hemogram disease, renal anemias such as low leukocyte due to a variety of causes, and make the exchange liquid of blood purification and the equilibrated fluid-supplement therapy of water salt etc.
(2) immune human plasma natural constituents preparation series:
With the method for sieving and the step of human normal plasma natural constituents preparation series, the serial component of acquisition is also corresponding fully with preparation.Just IgG component preparation wherein is specific immune globulin (IgG) preparation, is used for specific passive immunity's control of corresponding disease.
(3) immune serum immunoglobulin (goods) raw material components:
Two kinds of ultrafilter membranes with molecular cut off 70～150KD and 160～300KD, respectively deactivation immune serum material is sieved separation, obtain molecular weight and the close component of relevant animal IgG molecular weight (about 156KD), be the raw material components of immune serum immunoglobulin product, be prepared into corresponding goods for further refining purification.It provides the raw material components through inactivation of virus through preliminary purification for preparation animal immune globulin purification goods, and the refining processing and manufacturing of being more convenient for goes out corresponding immunoglobulin product, helps to improve the output and the quality of Related product.
(4) destination protein (goods) raw material components of biotechnology generation:
Size according to the protein molecular weight of purpose, select the molecular cut off two kind ultrafilter membranes bigger and more smaller for use than destination protein molecular weight, respectively the deactivation material is sieved separation, can obtain the destination protein component, be destination protein (goods) raw material components, be prepared into corresponding destination protein goods for being further purified, provide through preliminary purification, through the raw material components of inactivation of virus, be convenient to further refining purification and be prepared into the corresponding target protein product.
Preparation and/or material that above-mentioned (one), (two), (three), (four) make can be liquid dosage form or freeze-dried formulation, also all are simultaneously that confession is the raw material components of high-purity preparation with the additive method purification further.
Claims (8)
1, the plasma component preparation series of inactivation of virus/ultrafilter membrane segmentation sieve method preparation is characterized in that the preparation method that its process is following and the preparation series of preparation and/or raw material components:
Get raw blood plasma, remove virus filtration and the screening of 60-10KD ultrafilter membrane is filtered through clarification filtration step by step, nanometer film, acquisition surpass filtrate, or repeat aforesaid again except that virus filtration and ultrafilter membrane screening filtration, and the filtrate of ultra-filtration repeatedly that obtains through aseptic filtration, is the small molecule component preparation;
The ultrafiltration trapped fluid that obtains, or get raw blood plasma, again through following steps:
1) add the exogenesis protective agent,, carry out the damp heat inactivating more than 5-15 hour at 60 ℃ ± 20 ℃, or after lyophilizing, 80 ℃ ± 20 ℃ xeothermic deactivations of carrying out more than 50-100 hour;
2) carry out step by step clarification filtration and remove virus filtration with one group of different microporous membrane of membrane aperture, obtain material, title deactivation material through inactivation of virus/removals through nanometer film;
3) deactivation material promptly obtains the whole plasm component without the ultrafilter membrane screening; The deactivation material separates through ultrafilter membrane segmentation screening, obtains each serial component;
4) carry out ultrafiltration dialysis and ultrafiltration and concentration with dialysis with ultrafilter membrane, to obtain standard compliant serial component preparation;
5) carry out aseptic filtration with 0.20-0.22 μ m microporous membrane, obtain the plasma component preparation series and the raw material components of no any microbial activity, apyrogeneity matter, comprise the liquid dosage form and the freeze-dried formulation of micromolecule, middle molecule, macromole, macromolecule, comprehensive, whole plasm component preparation and raw material components etc.
2, the plasma component preparation series of inactivation of virus according to claim 1/ultrafilter membrane segmentation sieve method preparation is characterized in that raw blood plasma comprises the destination protein solution that produces with ACD anticoagulant anticoagulant human normal plasma, immune human plasma, immune serum or blood plasma, biotechnology.
3, the plasma component preparation series for preparing according to claim 1,2 described inactivation of virus/ultrafilter membrane segmentation sieve method, it is characterized in that human normal plasma, the relevant deactivation material of immune human plasma being sieved separation, obtain high molecular component preparation and large, medium and small molecular combinations component preparation or material with the ultrafilter membrane of molecular cut off 160～300KD.
4, the plasma component preparation series of inactivation of virus according to claim 3/ultrafilter membrane segmentation sieve method preparation, it is characterized in that large, medium and small molecular combinations component preparation or material being sieved separation with the ultrafilter membrane of molecular cut off 70～150KD, obtain the macromolecular components preparation and in, micromolecule combination partner preparation or material.
5, the plasma component preparation series of inactivation of virus according to claim 4/ultrafilter membrane segmentation sieve method preparation, it is characterized in that ultrafilter membrane centering, micromolecule combination partner preparation or material with molecular cut off 60-10KD sieve separation, molecular components preparation and small molecule component preparation in the acquisition.
6, the plasma component preparation series for preparing according to claim 1,2 described inactivation of virus/ultrafilter membrane segmentation sieve method, it is characterized in that two kinds of ultrafilter membranes with molecular cut off 70～150KD and 160～300KD, respectively deactivation immune serum material is sieved separation, obtain molecular weight immunoglobulin (IgG) component close with immune globulin (IgG) molecular weight (about 156KD) of relevant animal.
7, the plasma component preparation series for preparing according to claim 1,2 described inactivation of virus/ultrafilter membrane segmentation sieve method, it is characterized in that two kinds of ultrafilter membranes selecting for use molecular cut off bigger and more smaller than destination protein molecular weight, the destination protein component associated materials that biotechnology is produced sieves separation respectively, obtains the destination protein component.
8, the plasma component preparation series for preparing according to claim 1,2 described inactivation of virus/ultrafilter membrane segmentation sieve method; it is characterized in that saving from damage in the raw blood plasma on the basis of components such as self inherent native albumin; add and exogenously separate with the ultrafilter membrane segmentation about the laggard line correlation inactivation treatment of protective agent; the various plasma component preparation series that obtain are or/and confession is the raw material components of high-purity preparation with the additive method purification further.
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