JPS61280866A - Separation of human blood coagulating third factor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention (a) Technical Field of the Invention The present invention relates to human plasma blood coagulation factor Vl (hereinafter referred to as factor 1).
The present invention relates to a method for purifying factor 1:C from a concentrate of C).

(b) Prior Art For several decades, factor 1:C has been of great interest because of its usefulness in the treatment of hemophilia A.

The first factor in the body or in the circulatory system is composed of two different proteins [transport protein (von Williplan) 4
Factor Von Willibrand fac
It is known to be a large molecule complex consisting of factor 1 (C) and factor 1 (C), which are non-covalently linked to C or).

The transport protein reacts with platelets in a manner that primarily causes hemostasis and is usually associated with factor 1 R [ristocetin cofactor or WonWillib].
rand) antigen]. Patients with hemophilia A, they suffer from classic hemophilia and have active factor 1:C
and requires administration of factor 1:C to overcome bleeding.

Factor 1 (also known as antihemophilic factor A or AHF) is available medically as a lyophilized, sterilized dry powder that can be dissolved in sterile distilled water or sterile saline to treat hemophilic A: Provided for administration to patients.

Preparation of a plasma concentrate consisting of a blood coagulation factor complex or a portion of other plasma components containing the complex for clinical use requires many processing steps. The concentrate is essential for hemophilia A patients, but unfortunately its specific activity is low and therefore large doses are required in patients for the treatment of hemophilia A. be. Many methods have been developed for preparing such concentrates of high specific activity. Unfortunately, many of these methods have very low recovery rates and are expensive. Due to the low recovery and high cost of the process, these methods have not been utilized on a commercial scale to produce factor 1 concentrates with high activity.

Anderson et al.
al) U, 3.4G permit 3,920,625 is,
Twelve steps are disclosed for the separation of factor 1 (AHF) and factor B from plasma. The basic steps of this invention are:
It consists of adsorbing at least one of these factors onto a water-soluble gel carrier in a liquid phase, while the other factors remain in the supernatant.

U.S. Pat. A method for selectively removing fibrinogen from plasma solution is disclosed.

U by Johnson (J, H, Johnson)
, Sl? No. 4,397,841 treats plasma with two different water-insoluble cross-linked polyelectropolymers in the presence of exogenous heparin and separates factor 1 concentrate through successive adsorption steps. Discloses a method to image. In the first adsorption step, the factor I complex is removed from the supernatant. In the second adsorption step, the first factor is adsorbed.

After separation of the adsorbent from the supernatant, it is recovered from the adsorbent.

Recovery of 40-70% of raw material first factor has been reported.

Birsher et al. (E, G, Birger Blomb
U.S. Patent No. 4,348 by ack at al.
, 315 dissolved the impure stage factor complex in a solution containing at least 1.5 M glycine with a pH of about 6.3-7.8 and heated the resulting mixture at 15°C to 40°C. The present invention discloses a process for purifying and/or concentrating the Factor 1 complex by maintaining the Factor 1 complex at a temperature of .

U.S. Pat. No. 3,637,489 to W. Hallor discloses a process for separating blood components using stereochromatography. Hara processes plasma proteins by gel filtration, so-called gel chromatography, stereochromatography, and exclusion (e.g.
Xc1u810n) Discloses that it is known to be separated by means such as chromatography or gel filtration chromatography. Harrah further disclosed a method for fractionating plasma proteins on a laboratory scale, but it could not be used on a large scale and especially to purify the product to a point of medical application.

U, S, by Hurler (W, Hal'ler)
Patent No. 3,657,116 describes a method for fractionating plasma or serum using column chromatography packed with spherical glass particles.
A method for separating blood components is disclosed.

U.S. Pat.

Philip J, Fay at a
``Purification and characteristics of highly purified major factor 1 consisting of a single-type polypeptide chain'' (Proc, NatL) et al.
Acad.

Sci., Vol. 79, Page 7200 7
204. (December, 1982) discloses a method for purifying Ozutsu factor from plasma using calcium dissociation and different size and charge chromatography. Otsutsu■ factor is
It is purified over 350,000 times (vs. plasma) from commercially available factor 1 concentrates with a final recovery of 13%. What is the process?

Consists of 7 steps, special precipitation treatment, Bigel A-1
Fractionation on a 5M column, ammonium sulfate dialysis, calcium ion dissociation, chromatography on a Sepharose CL-4 column, dialysis, and finally chromatography fractionation on a QAK cellulose column.

Bioland Price List, T, (1984
January 1:) 94-99) Biogel TSK columns are made of polycationic or neutral polymers such as polyacrylamide (PAM), polyethylene oxide.

It is disclosed that the method is suitable for separating polyvinyl alcohol (PV'A) and the like. Biogel TSK column is
Filled with hydroxylated polyether derivative.

T, W, bar y, etc. are "HPLC peptides and proteins"
Am8riaan Laboratory, Voh
14 (10), October, 1982゜Bage 1,
Discloses the utility of high performance liquid chromatography in separating peptiR and proteins.

(c) Summary of the Invention The present invention provides a relatively simple size separation method for high-speed separation of human blood coagulation factor 1 from plasma preparations containing factor 1 and contaminants consisting of high-molecular and low-molecular substances.
) chromatography.

The present invention consists of the following steps.

(a) Preparing a buffered aqueous composition of a plasma product.

(A) Porous high performance liquid chromatography column (H
PLC column) to separate low molecular weight components from the buffered aqueous solution.

The column is a mechanically rigid support whose particle size is approximately 1
3 to about 35 microns. The particles are approximately 500-2
000 angstrom diameter and a dead volume of about 1.0 to about 1.8 pores. The column is eluted with an aqueous eluent, and a fraction eluted from the column containing a first factor substantially free of low molecular weight components and a high molecular weight component is selected to obtain a second water-soluble component.・It will be collected.

The second water-soluble component contains the first factor with excellent purity. The purity of factor 1 is increased 100 times. With normal methods, the luster increases by only about twice as much.

Factor 1 and Factor 1 200 specific activity in units of ~200 of protein is meant as a measure of purity. Large differences in specific activity are observed for the different elution fractions Factor 1 and Factor 1:C formulations. The difference in specific activity between the preparations of Factor 1 and Factor 1:C originates from the proteolytic enzyme activity of protease contained in impurities consisting of low molecular weight components and high molecular weight components.

Impurities include proteases that attack factor 1 and factor 1:C and inactivate coagulation factors. The impurities in each sample of Factor 1 and Factor 1:C have different protease activities.

Thus, each preparation of factor 1 and factor 1:C purified by the process of the present invention has a difference in the pre-purification ratio depending on the degree of proteolytic attack on factor 1 and factor 1:C by the protease in the impurity. They may have different ratios of activity and purified diameter specific activity.

Purification is carried out as quickly as possible to minimize attack on factor 1 by proteases in impurities. Plasma preparations containing factor 1 are lyophilized, kept at low temperatures, and maintained in a hydrophobic state to minimize protease activity until buffering of the aqueous solution. However, even with such precautions, protease attack during chromatography and concentration of collected fractions cannot be completely avoided.

The first factor is further dissociated into low molecular weight factor 1:C to remove high molecular weight components (impurities),
High molecular weight components and factor 1: C are separated by high speed size fractionation chromatography. The process consists of the following steps: (a) Dissociating Factor 1 to Factor 1:C in a buffer. That is, the salt concentration is at least 0.2 MVCi! to yield the second aqueous composition! Let's get everything in order.

(b) The high molecular weight components are then separated from the second aqueous composition by high performance column chromatography. The column is packed with a 9-pore mechanically rigid support, the particle size of the support being about 13 to about 10 microns. The particles have pores of about 500 to about 2000 angstroms in diameter;
The dead volume is approximately 1.0 to 1.8 Ti1l/l1.

The column is developed with an aqueous eluent to separate the eluted fraction from the column containing Factor 1:C substantially free of high molecular weight components.

The second aqueous solution composition obtained by the first chromatographic separation and purification (which is a recovered fraction containing the macromolecular component and the first factor substantially free of low molecular components) is used to remove the macromolecular components. A second purification treatment can be performed. The second aqueous composition obtained from the first chromatographic purification can also be directly subjected to the second purification treatment described above. Preferably, the second aqueous composition is concentrated before further purification. Concentration is carried out by means conventional to those skilled in the art. For example, dialysis, freeze-drying, evaporation, etc. Preferably.

Dialysis is performed to avoid concentration of buffers and salts.

The most commercial method of preparing factor 1 is from human plasma by proprietary processes such as cryoprecipitation. This method requires approximately 1 unit of powder/■
Prepare a lyophilized factor powder with a specific activity of . The specific activity of pure factor 1 is unknown.

It is thought to be more than 5,000 units. Commercial products of factor 1 concentrates include high molecular weight components (contaminants) and low molecular weight components (contaminants). The nature of these contaminants is not fully known, but they include fibrinogen, 7-on-Willyiprande protein, transport proteins, other blood clotting factors, and proteases.

The process of the present invention advantageously utilizes the molecular weight difference between Factor 1 and the low molecular weight component and the molecular weight difference between the dissociated Factor VIII, Factor 1:C1 macromolecular component.

The molecular weight of Factor 1 was reported to be 20,000,000 from i, o o o, o o o. The first factor is a low molecular weight type first factor in a high salt concentration, for example, 3MCcLC12 solution.
Factor: Dissociates into C. The molecular weight of the first factor: C is approximately 0.
9 x 105 to about 3. It has been reported that OX 10'[F'ay et al.].

In the first purification process, low molecular weight components (impurities) [this has a length of about 1100n or less di・m@n51on]
(which is approximately equivalent to a proteinaceous substance having a molecular weight of less than about 1,000,000)] is separated from the molecular weight components and the polymeric cylinder factor. In the second purification step, factor 1 is dissociated into low molecular weight forms under high salt concentration conditions.

and by a second chromatography about 1100n
length of more than aimansion) (this is about 1
,000,000 or more). 1
The method of the present invention allows approximately 100-fold or more purification of factor 1.

It is believed that when factor 1: C is depleted under high salt conditions, it recombines in the presence of Von-Willibrand factor. High salt concentrations can be reduced by addition of water, buffering or unbuffering, or preferably dialysis.

Contaminants associated with factor 1 are proteins, carbohydrates, lipids and mixtures thereof. These components differ greatly from low molecular weight components or high molecular weight components.
e ), they are separated from the first factor by current methods. If the first factor is bacteria, fungi (F'
ungi), viruses and/or these fragments that have a large difference from high molecular weight components (contaminants), such high molecular weight contaminants are removed from the first factor: C to the second step. separated by

(D) Detailed Description The present invention is directed to extracting Factor 1: C from a plasma preparation containing Factor 1, high molecular weight components (contaminants), and low molecular weight components (contaminants) by high-speed size fractionation chromatography consisting of the following steps. Regarding the method of separating: factor 1, a soluble and physiologically compatible inorganic salt concentration (approximately 0.0%) containing a high molecular weight component and a low molecular weight component
07 to about 0.3 M) and pHc about 5.5 to about 8.0)
A buffered aqueous composition is prepared consisting of a plasma product having a .

<b> Next, the buffered aqueous solution composition is subjected to high performance liquid column chromatography to separate low molecular weight components. The column is packed with a carrier having a particle size of about 13 to about 100 microns, with particles of about 500 to about 200 microns.
It has pores with an actual diameter of 0 angstroms, and the pore shape is about 1.0 to about 1.819/f1m. Elution is performed at a soluble physiological inorganic salt concentration (about 0.07 to about 0.3
M) with a buffered aqueous eluent having a pH of about 5.5 to about 8.0. Next, the high molecular weight component that is substantially free of the first factor and the low molecular weight component is separated and recovered to obtain a second aqueous solution composition.

If desired, the purified factor No. 1 composition is separated from the high molecular weight components by the following steps: (a) the second aqueous composition is concentrated into a third aqueous composition containing the factor No. 1 and the high molecular weight components; An aqueous composition is obtained.

The third aqueous composition has a protein concentration at the solubilization limit of Factor 1 and the high molecular weight component, preferably from about 0.1 to about 2005 M of one composition! Protein'X/l.

(h) Factor 1 in the third aqueous solution composition is then dissociated into a lower molecular weight form by addition of a soluble physiological salt under conditions of a salt cation concentration of about 0.2 M or more, and A composition is prepared.

(c) A high molecular weight component having a molecular weight at least equal to the molecular weight of the first factor is separated from the fourth aqueous solution composition by pore high performance liquid column chromatography. The column support has a particle size of about 13 to about 25 microns, and the particles have pores with a real diameter of about 500 to about 2000 angstroms.

The pore size is approximately 1.0 to 1.8 ml/I. Elution is performed at a soluble physiological inorganic salt concentration (about 0.07 to about 0.3
M) with a buffered aqueous eluent having a pH of about 5.5 to about 8.0. Then, both fractions containing dissociated factor 1:C and not substantially containing the high molecular weight fraction are recovered from the eluate.

Plasma raw materials include human, bovine, or pig plasma from 2 people, crude bovine or pig plasma from 9 people, and bovine or pig serum.

Crude serum from humans, cows, or pigs is used. A preferred plasma source is factor 1 concentrate, particularly lyophilized factor powder. If a commercialized factor 1 concentrate is used as a raw material, it is dissolved in sterile distilled water or sterile buffer and used as a buffered solution composition formulation. The buffer used is a conventional biochemical buffer, especially one with a maximum buffering capacity of pH 5.
, 5 to 8.0. A preferred buffer is imidazole hydrochloride. As the soluble physiological inorganic chlorine salt, a conventional physiological inorganic chlorine salt that does not react with or denature proteins is used. For example, NaC
1,0g C12, LLC12T KCl, bc12, etc., preferably NaCl is used. The ionic concentration of the chloride salt is maintained below 0.5 M in a composition, preferably between about 0.07 and 0.3 M, more preferably about 0.
．． It is 15M.

Chromatography columns are subjected to high pressures for high performance liquid chromatography. Therefore, the column is made of a strong inert metal such as stainless steel.

The column support is inert with the aqueous solutions used in chromatography and is non-toxic and non-reactive with proteins such as Factor 1 and Factor 1:C.

It is also necessary that it be mechanically rigid and not undergo any deformation even under the high pressure of high performance liquid chromatography. Hydroxylated polyether carrier such as Bio-Rad Biogel T
SK carrier (Toyo Soda Nihon) is preferably used.

The TSK carrier contains -CH2CHOHCH20 as a component.
It was reported to be a hydrophilic polymer gel with - substituents [Hashimoto et al., J. Polym.

Sci, Polym, Phys, EalVol,
16 (1978).

1789-1800). The particle size of the carrier is important;
The size should be about 13 to 10 microns to allow adequate clearance distance between the particles to allow proteins and other impurities to pass through the column. The pores of the particles are substantially from about 500 to about 2000 angstroms, preferably about 1
It has a diameter of 000 angstroms. Pore size is important. The reason is that the pores must be large enough to allow the first factor and high molecular weight components to rapidly elute and separate from the column during the initial chromatographic separation, while slowing the elution of the lower molecular weight components. Conversely, the pores contain dissociated VIII
The elution of the factor is delayed to such an extent that the high molecular weight component is rapidly eluted from the column during the second chromatographic separation, thus inhibiting the separation of the high molecular weight component and dissociated Factor VIII. Leprosy cannot do less. The pore volume of the carrier is about 1.0 to about 1.8f
fi4/g and 1% preferably from about 1.2 to about 1.
It has a pore volume of 5d/lit.

The packing of the small particle carrier into the column must be such that the particles are closely packed together to prevent the formation of detours. Bypassing during chromatography adversely affects the separation. Preferably, the column is S'1urr7
) (muddy fluid) under high pressure e.g. 2000 psi
filled with.

The aspect ratio of the column, ie the ratio of the depth to the inner diameter of the column, is preferably between about 10 and 10.

Columns with lower aspect ratios have poorer resolution than columns with higher aspect ratios. The choice of column aspect ratio depends on the degree of separation desired and the time of separation.

The pressure used in the separation step is not important per se.

Sufficient pressure is used to elute at the desired flow rate. There is no minimum limit to the amount of pressure used, and pressures close to atmospheric pressure are used. The mechanical limits of the equipment and the mechanical strength of the support determine the upper pressure limit that can be used during the process. 2500p
The pressure is more than si.

pressures of about 100 to about 2000 psi, preferably 200 to 1000 psi.
A pressure of psi is used. The linear flow rate of elution from the column usually depends on the degree of separation desired and the separation time. For the first chromatographic separation of the present invention, a linear flow rate of about 0.5 to 105 M min was found to be sufficient, and for the second chromatographic separation, about 1.2 to 5 M min. A linear flow rate through the column of .0 cIrL/min was found to be sufficient.

After the first chromatographic separation, the total volume of fractions containing Factor 1 was significantly larger than the aqueous composition used for the first separation. Although it is not necessarily necessary,
Preferably the collected fraction or second aqueous composition comprises:
For example, it is concentrated by conventional means such as dialysis or freeze-drying.

Preferably, the second aqueous composition is concentrated 5 to 10 times to form the third aqueous composition. That's the third
is concentrated to maximum solubility of Factor 1 and high molecular weight components in an aqueous composition, preferably from about 0.1 to about 20 Of! of the composition. Protein/l.

Most preferably about 1005M! protein/l. After initial chromatographic separation, factor 1 is extracted from water-soluble physiological salts such as NaCl, potassium nitrate.

Treated with CaCl, preferably CaCl2. The concentration is sufficient to dissociate factor 1 into lower molecular forms. Any soluble physiological salt can be used in column packing or processing steps as long as it does not deleteriously affect Factor 1, Factor 1:C. Usually, preferably CaC
l2 is used for dissociation.

The salt concentration of the water-soluble composition containing factor 1: C is approximately 0.2M.
When adjusted to preferably about 0.25 to about 0.5M, more preferably about 0.3 to about 0.35M,
The factors are approximately 0.9 and 3. It dissociates into lower molecular forms with molecular weights between OX105 and OX105.

Compositions comprising dissociated factor VIII preferably contain
A chromatography column that is the same or substantially the same as a chromatography column. Second
The buffered aqueous eluent used in the first chromatographic separation was substantially the same as that used in the first chromatographic separation. However, the salt concentration is approximately 0,
25 to about 0.5 M preferably about 0.3 to about 0.3
It is 5M. The linear flow rate of the twentieth column is preferably slower than the flow rate of the first column to facilitate separation of dissociated Factor VIII from high molecular weight components. In the second purification process.

Dissociated factor VIII is small enough to pass through the pores of a packed column. Smell to delay the elution of dissociated factor VIII. High molecular weight components are too large to pass through the pores and are eluted from the column faster, thus achieving the desired separation.

The eluate from the chromatography column passes through a proteinaceous substance detector. The proteinaceous substance of factor 1 is 2
It strongly absorbs light with wavelengths between 54 lumen and 320 nm.

The absorption properties of the eluted fraction from the column can be monitored at any wavelength between 2567LrL and 320rLm using a conventional spectral photometer. 1st
Both fractions containing the factor are collected and collected. The collected fractions are concentrated using conventional methods such as dialysis, lyophilization, etc. The Factor 1:C obtained by the second chromatographic separation is concentrated, reconstituted, or subjected to further processing for commercial or study of the structural properties of human, bovine, and porcine Factor 1. .

[Brief explanation of the drawing]

According to FIG. 1, a buffered aqueous solution composition 10 of plasma components consisting of factor 1 high molecular weight components (contaminants) and low molecular weight components (contaminants) is introduced into syringe 14. Buffered eluate 11 is introduced or siphoned into pump 12 and pumped into guide column 16 through tube 13A, three-way pulp 15A and tube 13B. Buffered composition 10 is pumped into guard column 16. That is, the pulp 15A connected to the pipe 17B is opened, and the pipe 1
Close the pulp 15A leading to 3A and open the pulp 15B to pump the eluate 11 from the pump 12 into the syringe 14 through the tube 17A and transfer the composition IO to the tube 17B.
, is injected into guard column 16 via tube 15A and tube 13B. Eluate 11 and composition 10 have a concentration of about 15 to about 3
Guard column 16.000 psi at elevated pressure.
High performance liquid chromatography column 18. Detector,
It is pumped into and through fraction collector 26. Guard column 16 acts as a filter, collecting materials that may obstruct or plug high performance liquid chromatography column 18. The filtered aqueous buffered composition is transferred under pressure from column 16 to high performance liquid chromatography column 18 for subjecting it to a first chromatographic separation to separate factor 1 and low molecular weight components from high molecular weight components. will be expanded. The eluate 22 from the column 28 passes through a chromatophotometer detector 24 where proteinaceous substances can be detected. It is collected in a conventional fraction collector 26. Low molecular weight components with lengths below 1100n enter the pores of the column packing and their elution is delayed. The first factor and the high molecular weight components are too large to enter the pores and are eluted quickly from the column, resulting in separation from the low molecular weight components. Fractions containing low molecular weight components are discarded. The fraction containing the high molecular weight component and factor 1, which elutes before the low molecular weight component, is fractionated by a 7 lactation collector 26 and the 0 fraction 28, collected as fraction 28, is described below. The concentrated and buffered aqueous compositions are prepared in a conventional manner. And composition 30 is. %CaCJ2 as described in dissociation step 32, factor 1 is dissociated into lower molecular weight forms. A dissociated factor 1 composition containing a high molecular weight component is injected into the injector through the tube. Buffered eluate 35 containing the same concentration of CaCl2 as the dissociated factor VIII composition is
siphoned or introduced into pump 36. It is then pumped into the guard column 40 through the three-way pulp 3gA into tubes 37A and 37B. The dissociated factor VIII composition is injected as a Slug. That is, open the pulp 38A leading to the pipe 39B, and open the pulp 38A leading to the pipe 39B.
Close pulp 3gA leading to 7A. and pump 36
from the tube 39A and pulp 38B into the syringe 34,
and syringe Akara tube 39B, pulp 3gA and tube 3
Open pulp 35B to pump the eluate through 7B and into the guard column chest. The syringe has a relatively small diameter joining one end of tube 39 and the other end of tube 39B, and the syringe bore is connected to the tube for filling the tube by injection of the dissociated factor VIII composition. connected. Guard column 40 is the same as guard column 16,
It acts as a filter to remove components that plug or obstruct the high performance fluid chromatography column 8. This column is substantially identical to column 18. Dissociative type V
The factor III composition is chromatographically treated on column C to separate dissociated factor VIII from high molecular weight components. Factor 1: C is a low molecular weight form of the primary solid, which can pass through the pores of a packed column. This substance then elutes from column C after the high molecular weight components that are too large to pass through the pores of the packed column elute. Column C is developed with buffered eluent. That is, from pump 36 to the column core, it passes through tube 39A, pulp 38B, syringe 34% tube 39B% pulp 3gA and tube 37B, or tube 37A, tube 3gA and tube 3.
It is pumped by passing through 7B. The eluate from column C passes through a Siktro 7 otometer detector 46 and is collected in a 7 lux collector. The detector 46 can detect protein-like components. Fractions containing Factor 1:C are collected, mixed, and subjected to further processing. For example, it is prepared into a commercial Factor 1:C concentrate for medical or research use. Other fractions, for example those containing high molecular weight components, can also be used for research purposes. The support particles of the GART 0 column are short columns consisting of particles of the same composition and of the same particle size or larger size as those of the high performance liquid chromatography column. No separations are performed on guard columns. The function of the guard column is to remove components that would interfere with the interstitial spaces between the particles of the high performance liquid chromatography column. Examples of the present invention are shown below, but these are not intended to limit the present invention. (f) Examples (a) Apparatus and materials The liquid chromatography used in the following examples consists of the following. Model 6000A pump, type UK
6 note size (includes 10 samples), Model 450 variable length detector. All of these are Water
Aasociates, Milfora, Mar
It is made by yland. A semi-prepared TSK 5ooo pw column (2,5X 60s 9 particle size 17 microns) was used as the separation column, and a TSK 6000PW column A
(2,5X 7.5c!rL) was used as a guard column. These columns are manufactured by Toyo Soda. Buffered aqueous solution is high performance liquid chromatography (HPLC)
Prepared to the extent of. New Jersey's J, T, Baker, Ph
Obtained from illipsbvrg. All chemicals used in the experiments were of reagent or analytical grade. Person 125 Factor 1: C, J, L, Moake
(BLOOD, 61 (1983) 116
3) Prepared according to. <b> Determination of protein Protein concentration was determined using the fluorometric analysis method of P, Bohlen, etc. (ARCH, BIOCHEJ B Engineering 0PH
Y.S. 155 (1973) 213) or M, Bra.
dford method [ANAI, YT engineering CAL B engineering OC
Measured by Mi M., 72 (1976) 248). (c) Ij solid analysis The activity of factor 1:C of complex factor 1 and dissociated factor VIII was measured by the following method. ■ The method of L. Tocanthis et al. (BLOODC
OAGULAT ON ENGINE HEMORRAGE A
NDTEROMBO8ENGS M胛HODS OF'
5TUDY New York, Grune & 5
Tratton, 1965) Two-step thromboflastin generation test time method. ■ The method of L, Tocantis, etc.
EMO3TASIS Philielphia, W, B
, 5unders, 1971.111page)
...One-step activation tromposus sustaining time test method. In both methods, plasma obtained directly from hemophilia A patients or Geor
Hemophilia A plasma purchased from King Biomedica 1 was used as a substrate. Analysis of all first factors: C
te for Biological Stanclar
ds and Control, London,
Human blood coagulation factor (805) obtained from England
M556) The first factor calculated against the standard was performed on the in-house standard. 171oo, Ng O2't
Double dissociation at oO and yO was tested for each sample and standard except for the column fractions. In some cases. The Kadan factor:C activity in the column fraction is
Determined photometrically using the umCOATEST As5ay Kit. This test measured factor 1:C activity based on the ability to convert factor X to factor Xα, and yielded results identical to those obtained using coagulation analysis. (d) Purification of Factor 1: 10 bottles of commercially available Ton Willibra
The complex with ndl"actor) concentrate (trade name Prophylate, manufactured by Alpha) was dissolved in 3% wLl of water and pooled. Buffer exchange of the pooled material was performed. Pooled onto a 5 ephadax G-25 column (Pharmacia) Fill the sample and then add buffer A (50
mM imidazole hydrochloride, (59mM NaCl, pH 7
, 0, 0,02 tindeium azide). 28
All detectable protein eluted in the void volume fraction, as measured by absorbance at 03m, was essentially pooled. 5 on an aphadex G-25 column. Twenty filtered factor 1 concentrates were added to T S K 6
000pw Gart 9 column (2,5X 7.5 cyt
) pre-prepared Toyo Soda TSK 500 combined with
0 PW column (2,5X 60cIt) to make sure. Then, about 8 ml of buffer solution AK was passed through the column.
It was eluted at a flow rate of 8.5 m7 min, equal to a linear elution rate of 7 min. Fractions were collected at incremental intervals, using a G11ford M
With Odel 2600 optical measuring machine. The absorbance was analyzed by measuring the absorbance of 254iyx. A divisible fraction (A11quots) from each half was analyzed for factor 1:C activity. The fractions with blood coagulation (procoagulant) activity are pooled. Fixed Aquaciae IF (Behring-Ca
lbiocham) and concentrated. The concentrated protein sample was then diluted with buffer A K 3-5.
Add t amount of M CcLC12 solution, CaCl2
The complex type factor was dissociated into its lower molecular weight form. This material is then subjected to the aforementioned HPL
C column and 0.35 M CaCl
2 Buffer A with a concentration of 4.5 mu/min (d)
It was eluted at a flow rate of approximately 3 cIL/min (equivalent to a linear elution rate of 3 cIL/min) through the ram. Separated fractions were collected in silylated glass vessels and at 1 minute intervals. The eluate was 280
The absorbance at nm was continuously analyzed. Each fraction, at least 8
diluted twice. The active fractions are pooled and added to buffer A or buffer A containing 2516 volumes of glycerol.
Concentrated by vacuum dialysis. (6) Chromatographic separation of commercial cylinder factor (Vcn Willibr) of approximately 1.511
complex with ancl factor) concentrate (manufactured by Alpha:
Two vials of Profilate (trade name) were filtered through a Sephadex G25 perforated column. The filtered factor 1 concentrate was mixed into a buffered solution and
K 6000 PW guard collar A (2.5x 7.5
CI! L) [Toyo 5oda TSK
Injected onto a 5000PW column (2,5x60crIL). and 8 equal to a column linear flow rate of about 8 cm/min.
．． Buffer A was developed at a flow rate of 5 Ml/min. Individual fractions were collected at 1 minute intervals. The absorbance of each fraction is
Determined by spectrophotometer at 254f47B. Divisible fractions from each fraction eluting after the void (vol) quinine were diluted 400 times. In the TGT analysis in which this dissociation is determined, several seconds are required for clot formation to take place. The activity of each fraction is inversely proportional to the clotting time of this assay,
Factor: 17 as it can be said to be linearly related to C activity.
Expressed as clotting time. The arrow at the top of Figure 2 indicates the standard compound of a specific molecular weight under the same conditions. 1 shows an apparatus consisting of columns. Standard compounds are thyroglobulin, 7-erythin, and bovine serum albumin. Beta-lactoglobulin, guanosine. As shown in FIG.
It was separated from the lower molecular weight components present in commercial factor 1 formulations by high speed size exclusion chromatography on a PW column. It is known that in ionic strength buffers, Factor 1 20 binds to van Wnlibrand factor and exists as a high molecular weight complex. Factor 1 was found to be eluted from the column in a volume of void 9 (Ma05Md) with a molecular weight fraction in excess of 2×10 2 (see graph A in FIG. 1). The separation is completed within 25 minutes and approximately 90 fractions of Factor 1:C activity injected onto the column is collected in a pool of active fractions that are eluted. The degree of purification of this step was 20-40 times. The isolated factor 1 had the ability to activate thrombin. Polyethylene glycol (4000) precipitated or fixed aq
A subsequent concentration step by dialysis against uacle II resulted in a ~45% decrease in activity. Most of the loss in Factor 1:C activity was thought to result from proteolytic attack on Factor 1 by proteases in impurities remaining in Factor 1 after purification. The concentration step takes place at room temperature (approximately 21° C.). Proteases are active at this temperature. Approximately 41 I of the commercial factor 1 concentrate was chromatographed by the column at one time. However, chromatograph 4-
Due to the size of the sample loop for the column, a total volume of IQmJ of fuel is injected at once. As a result, the actual amount of protein ciples injected into the column was limited by sample concentration and sample loop size. In some cases, several consecutive injections were necessary to purify the desired sample amount. Filtration of the Factor 1 concentrate prior to purification by the process of the invention is preferred. Less than 10 units of Factor 1:C activity of the Factor 1 concentrate was lost after filtration on a Sephadex 025 column. Complex type factor 2: C (factor 1) was dissociated from the WHO-ebrana factor under conditions of high concentration of CαC12. Approximately 10% of the factor 1 purified in the above example was concentrated and 0-35 M to dissociate factor 1.
In a composition having a concentration of CaC4. The composition was developed with the same high performance size fractionation liquid chromatography column as in the example above. The column was developed with buffer A with a ctzc72 concentration of 0.3 M at a rate of 311 Lt/min (equivalent to a linear flow rate of the column of approximately 3 cr! L/min). Individual fractions were collected at 1 minute intervals and the absorbance of each fraction was determined using Water Mo
Determined spectroscopically on a DEL 450 optical spectrometer at 28 [cotton]. void volume
All fractions eluted after are eluted with 0.02% weight of sodium azide and 0.05% weight of imidazole hydrochloric acid.
It was diluted 20 times with a pH 7.0 buffer containing M concentration. These buffered compositions were assayed for Factor 1:C activity as described in the Examples above. The arrows at the top of Figure 3 indicate standards with specific molecular weights. An apparatus consisting of columns under the same conditions is shown. The standards were the same as those used in the previous examples. No dissociation of oligomeric protein molecular weight markers was observed. Factor 1:C activity is determined by void volume (valdvo
xume), but as a more pronounced low molecular weight broad peak indicating that this factor was dissociated from a high molecular weight complex (see graph D in Figure 3). Factor 1: The peak of C activity is approximately 100
Corresponding to a molecular weight between 1,000 and 300,000, it was probably identified by 125-nee Fab-8DS polyacrylamide gel electrophoresis (described above).
It consisted of a substance with a molecular weight of 6 x 10 and some of its decomposition products. When this method was used to further purify Factor 1:C from high molecular weight materials separated by high performance size fractionation liquid chromatography under conditions of low ionic strength, a 2-3-fold increase in purity was achieved. Commonly seen. The recovery rate is 35% of the injected activity.
It was a lot. High molecular weight complexes containing Factor 1:C will not pass through high performance liquid chromatography columns packed with particles of diameter less than 10 microns. The advantages of the method of the invention are its rapid purification, simplicity and possibility of scale-up. The method of the invention is of great value for the treatment of hemophilia A patients and to the plasma fractionation industry. and) gel electrophoresis analysis factor 1: C, J, L, Moake 5upra
It was analyzed by the basic gel electrophoresis method described by K. 25 microliters of partially purified Factor 1:C was dissolved in 5mM diimpropylfluorophosphate (Aldriah), 95N
Engineering HU/-iludin, 367 kallikrein inhibitor U, 4/abrochi = y (Trasy'lol,
FBA Pharmaceutical Hz-York) and average molecular weight 4000
125 containing 3.2 volumes of polyethylene glycol
- Knee factor 1: CFab (total amount 13,000 cpm
) was incubated with η microliters. Incubation was for 90 min at 37°C followed by 30 min at 0°C.
It was done for a minute. The reaction mixture was warmed to room temperature, and this was combined with [0,18 M) Lis-HCI (pH 6,8), 6
% (W/V) 7 dei/l/sulf x-)
52 microliters of a solution containing (SDS), 15% (w/v) 2-1 captoethanol, and 3 oz (v/v glycerol) was added and incubated at 37°C with gentle agitation. After 30 minutes, the whole mixture was mixed with U, Laemmli, N
ATURE (Lonclon), 227 (1970
) 680 by electrophoresis on 5 polyacrylamide slab gels containing 0.11 SDS. Protein molecular weight markers were run in a lane of the slab gel adjacent to the lane containing the reaction mixture. After the run, the part of the gel slab containing the molecular weight markers was removed and injected with 25% (Vlv) Improper Tools, 10
% (V/7) Glacial acetic acid, sled, Te 0.15% (W/￥
) Stained for 2 hours with a solution containing Cooma 5sie Ble. Then 5% (V/V) methanol,
Destaining was performed with 10% (Vyt) glacial acetic acid. The relative mobility of individual markers in the gel was determined. The portion of the slab gel containing the reaction mixture sample was 2F% (Vl
'i) Improper tool, 2 in 10% (Vv) glacial acetic acid
Time fixed 1 then 704 (V7Y) ethanol, 10% (V/￥) glacial acetic acid, 5% (V
/V) Solution containing glycerol [soaked for 2 hours. dried and XAR-5XOmat l;'im (E
astmanICalak, Rochester,
New York) and KoaakX-OmatiQ
The regular intensifier screen was left at -70°C for 5 to 7 days. The relative mobility of Bant 9 observed in autoradiographs was determined and compared to protein molecular weight markers. (y) Characteristics of the molecular weight type of factor 1: C In order to determine the molecular weight type of the dissociated hot factor and procoagulant complex present in the high speed size fractionation liquid chromatography preparation, this substance was - Knee 1st
Solid child: CF'ab and inquie) L, 5DS-5
% polyacrylamide P gel electrophoresis and octoradiography. The biggest and most important bunt, i.e. 1st factor: C Shao + 125 - Engineering - 1st factor: CFa
b complex band. It is a broad band with a molecular weight range of 3.4 x 10' to 2.7 x 105. These were calculated as a molecular weight range of 2.9 X 105 to 2.2 X 105 after removing the 0.5 X 105 molecular weight of 125-I-factor 1: CFab. This molecular weight of approximately 2.6 x 105 was determined by P. J. Faye et al. and M., T. It is equivalent to the molecular weight of the intact form of blood coagulation factor 1:C precursor reported by Weimstein. This is a molecular weight type of factor 1 of 20, which is the main component in fresh human plasma. This type is produced by proteolytic cleavage of factor 1:C
Decreased after activation of the molecule. The low molecular weight form of Factor 1:C is also evident in the autoradiogram, which has a calculated value of 1
．． It has a molecular weight of I x 10 . The molecular type of factor 1 20 with this molecular weight is M, J, Wainst
Reported by Ein et al. (5upra) TIC. And this is due to proteolytic cleavage 2) 6 x 105
．． It is believed that it originates from the molecular weight. Other forms of Factor 1:C with molecular weights below 2.6 x 10 are known to exist, but are not observed on autoradiographs and have not been separated by high speed size fractionation liquid chromatography. It was suggested that it does not exist in high molecular weight complexes. (g) Summary of the invention Continuous high speed size fractionation chromatography. Factor: C (complex factor 1) concentrate can be reconstituted on a large scale using firstly under low-salt conditions and secondly under high-salt conditions for use in humans, cattle, and pigs. A method for rapidly and easily purifying blood coagulation protein factors. Chromatographic separations are carried out in high speed size fractionation chromatography columns packed with porous supports. The particle size of the carrier is approximately 13
The diameter of the pores is from about 500 to about 20 microns.
00 angstroms and the pore volume is about 1.0 to about 1.81111/l1. The first chromatographic separation is performed using a buffered aqueous solution as the eluent. Low molecular weight components (impurities) and high molecular weight components (impurities) are separated from the first factor. In the second chromatographic separation, Factor 1 was dissociated in a buffered aqueous solution containing calcium ions at a concentration of about 0.25 to about 0.45M. The second chromatography column is packed with the same support as the tenth column and eluted with a buffered aqueous solution containing calcium ions at a concentration of 0.25 to 0.45M. On a 2.5 x 60α column, 42% of the commercially available factor concentrate is purified within 2 hours. This process is. It can be easily scaled up. 4. Brief Description of the Drawings Figure 1 is one of the best flow sheets of the process of the present invention. FIG. 2 shows the elution and turn of a human plasma preparation in the first column chromatography of the present invention. A indicates the absorbance at 251, and B indicates the activity of factor 1:C. FIG. 3 shows the elution sequence of human plasma preparations in the first and 20th column chromatography of the present invention. C
is the absorbance of 280fim, and D is the procedural amendment dated February 73, 1985.

Claims (1)

[Claims] 1) Factor VIII coagulation protein (factor VIII) is separated by high-speed size fractionation chromatography consisting of the following steps.
Method for separating from a plasma fraction containing factor VIII, a high molecular weight component, and a low molecular weight component: (a) A buffered aqueous solution composition of a plasma fraction consisting of factor VIII, a high molecular weight component, and a low molecular weight component is prepared. (b) Injecting the buffered composition into a chromatography column consisting of a porous high performance liquid chromatography carrier to separate low molecular weight components from the composition. The carrier has a particle size of about 13 to about 35 microns;
00 to about 2000 angstroms in diameter. The pore volume is about 10 to about 1.8 ml/g. Elution is carried out by developing the chromatography column with a buffered eluent, and selecting the eluted fraction from the column containing factor VIII and high molecular weight components, which are substantially free of low molecular weight components, and separating the fractions from the column with a second water-soluble eluent. Obtain a composition. 2) The method of claim 1, wherein the buffered aqueous composition is a soluble physiological inorganic chlorine salt having a concentration of from about 0.07 to about 0.3M, and from about 0.025 to about 0. It consists of imidazole hydrochloric acid at a concentration of .075M. The buffered aqueous eluent is soluble, about 0.07 to about 0.0.
It consists of a physiological inorganic chloride salt with a concentration of 3M, imidazole hydrochloric acid with a concentration of about 0.025 to about 0.075M. The buffered composition and buffered eluent described above each have a pH of about 5.5 to about 8.0. 3) In the method of claim 2, the buffered aqueous solution composition and the buffered aqueous eluent are each soluble;
Physiological inorganic chlorine salt at a concentration of about 0.15M, about 0.05M
of imidazole hydrochloric acid, each having a pH of about 7.0. 4) The method of claim 1, wherein the high molecular weight component has a molecular weight at least equal to the molecular weight of Factor VIII and the low molecular weight component has a molecular weight not greater than about 1.0 x 10^6. 5) In the method of claim 1, the buffered aqueous composition is developed through the chromatography column at a linear rate of about 0.5 to 10 cm/min. 6) In the method of claim 1, the ratio of the carrier length of the chromatography column to the inner diameter of the chromatography column is about 10 to about 40. 7) In the method of claim 1, the buffered aqueous composition is eluted from the chromatography column at a pressure of about 100 to about 2000 psi. 8) The method of claim 1, wherein the buffered aqueous solution composition is at a pressure of about 200 to about 1000 psi.
Eluted from the chromatography column. 9) The method of claim 1, comprising the step of adding: (c) the concentration of the soluble salt in the second aqueous composition is about 0.
VII in the composition by adjusting it to 2M or more.
Factor I is dissociated into a low molecular weight form to obtain a third aqueous solution composition containing dissociated factor VIII and a high molecular weight component. (d) injecting the third composition into a chromatography column of a porous high performance liquid chromatography carrier;
High molecular weight components are separated from dissociated factor VIII in the composition. The carrier has a particle size of about 13 to about 35 microns and the particles have pores of about 500 to about 2000 Angstroms in diameter. The pore volume is about 1.0 to about 1.8 ml/
It is g. Development of the chromatography column is carried out with a second buffered aqueous eluent, which has a potential salt concentration of about 0.2M or greater. Then, an eluted fraction from the column containing dissociated factor VIII that does not substantially contain the high molecular weight component is collected. 10) The method of claim 9, wherein the third aqueous composition and the second buffered aqueous eluent are soluble, physiological inorganic chlorine having a concentration of about 0.07 to about 0.3M. salts, including imidazole hydrochloride having a concentration of about 0.025 to about 0.075M, each having a concentration of about 5.5 to about 8.0M.
It has a pH of 11) The method of claim 10, wherein the third aqueous composition and the second buffered aqueous eluent are soluble, physiological inorganic chloride salts at a concentration of about 0.15M;
0.05M concentration of imidazole hydrochloride, each containing approximately 7.0M of imidazole hydrochloride.
It has a pH of 0. 12) In the method of claim 9, the high molecular weight component has a molecular weight at least equal to the molecular weight of Factor VIII. 13) In the method of claim 9, the third aqueous composition is eluted from the chromatography column at a linear rate of about 1.2 to about 5.0 cm/min. 14) In the method of claim 9, the length of the porous carrier in the chromatography column of step (d) relative to the inner diameter of the chromatography column of step (d) is
From about 10 to about 40. 15) In the method of claim 9, the third water-soluble composition is eluted from the chromatography column of step (d) at a pressure of about 100 to about 2000 psi. 16) In the method of claim 9, the third aqueous composition is eluted from the chromatography column of step (d) at a pressure of about 200 to about 1000 psi. 17) The method of claim 1, wherein the porous high performance liquid chromatography support in the chromatography column has a particle size of about 15 to about 19 microns;
The pores had an average diameter of about 1000 angstroms. 18) The method of claim 9, wherein the porous high performance liquid chromatography support in the chromatography column has a particle size of about 15 to about 19, and the pores have an average diameter of about 1000 angstroms. Was. 19) Factor VIII blood coagulation protein (factor VIII), a high molecular weight component with a molecular weight at least equal to factor VIII;
A method for separating factor VIII from a plasma fraction containing a low molecular weight component with a molecular weight of 1.0×10^6 or less by high speed size fractionation chromatography comprising the following steps: (a) factor VIII; a high molecular weight component with a molecular weight at least equal to the molecular weight of Factor VIII, and 1.0 x 10^
A buffered aqueous composition of a plasma fraction containing low molecular weight components with a molecular weight of 6 or greater is prepared. The composition comprises a soluble physiological inorganic chlorine salt at a concentration of about 0.07 to about 0.3M, about 0.025 to about 0.07M.
It contains imidazole hydrochloric acid at a concentration of 5M and has a pH of about 5.5 to about 8.0. (b) injecting the buffered aqueous solution composition into a chromatography column of a porous high performance liquid chromatography support;
Separating low molecular weight components from the composition. The carrier is about 13
It has a particle size of about 35 microns, and the particles are about 5
00 to about 2000 angstroms in diameter, and the pore volume is about 1.0 to about 1.8 ml/gm. The elution includes a soluble physiological chloride salt at a concentration of about 0.07 to about 0.3M and imidazole hydrochloric acid at a concentration of about 0.025 to about 0.075M and about 5.5 to about 8.0M.
The column was developed with a buffered aqueous eluent having a pH of . Then, the eluted fraction from the column containing high molecular weight components and factor VIII substantially free of low molecular weight components was selected and fractionated to obtain a second aqueous solution composition. (c) Concentrating the second aqueous composition to obtain a third aqueous composition containing Factor VIII and a high molecular weight component. The third composition has a protein concentration of about 20 to about 200 g protein/l composition. (d) adding a soluble, physiological salt to the third aqueous solution composition and adjusting the salt concentration from about 0.25 to about 0.5 M to dissociate Factor 1 into a low molecular weight form; A fourth aqueous composition is prepared. (e) injecting the fourth aqueous composition into a column of a porous high performance liquid chromatography support to separate high molecular weight components from the fourth composition at least equal to the molecular weight of Factor VIII; the carrier has a particle size of about 13 to about 35 microns;
The particles have pores of about 500 to about 2000 Angstroms in diameter. Pore volume is about 1.0 to about 1.8 ml/gm. The elution is carried out at a pH of about 5.5 to 8.0 with a soluble physiological inorganic chloride salt at a concentration of about 0.07 to 0.3M, a soluble physiological inorganic salt at a concentration of about 0.25 to about 0.45M.
0 buffered aqueous eluent. Then, an eluted fraction from the column containing dissociated factor VIII substantially free of the high molecular weight component is collected. 20) The method of claim 19, wherein a buffered aqueous composition, a buffered aqueous eluent, a fourth aqueous composition,
and a second buffered aqueous solution eluent comprises a soluble physiological inorganic chlorine salt at a concentration of about 0.15M, imidazole hydrochloric acid at a concentration of about 0.05M, and each buffered aqueous solution composition, the first The pH of the buffered aqueous eluent, the fourth aqueous composition, and the second buffered aqueous eluent is about 7.0. 21) The method of claim 19, wherein the length of the porous support of the chromatography column and the internal diameter of the column are about 10 to about 40. 22) The method of claim 19, wherein the first aqueous composition is eluted from the first chromatography column at a linear rate of about 0.5 to about 10 cm/min. The fourth aqueous composition has a molecular weight of about 1.2 to about 5.0
It is eluted from the chromatography column at a linear rate of cm/min. 23) In the method of claim 22, the buffered aqueous composition is eluted from the chromatography column at a pressure of about 100 to about 2000 psi. The fourth aqueous composition is eluted from the chromatography column at a pressure of about 100 to about 2000 psi. 24) The method of claim 22, wherein the buffered aqueous composition is eluted from the chromatography column at a pressure between about 200 and about 1000 psi, and the fourth aqueous composition is eluted at a pressure between about 200 and about 1000 psi. It is eluted from the chromatography column at a pressure of 1000 psi. 25) The method of claim 18, wherein the support of the porous high performance liquid chromatography column has a diameter of about 15 to about 1
The particles have a particle size of 7 microns and the pores have an average diameter of about 1000 angstroms.