DE2037188A1 - Process for separating proteins - Google Patents

Description

PATINTANWXLTE
DR.-ING. H. FINCKE β münohen β. 2 7. JufMSTO

DIPL.-INQ. H. BOHR MM »* ™ ·, 3»

DIPL.-ING. 3. STAEQER
did »ret,» Hiti $

Neppe 8438 - Dr.K.

Institute Llorente, S.A. Madrid / Spain

"Procedure for the separation of proteins ¹

Priority July 26, 1969 - Spain

The firing is based on a method of separation of highly pure proteins from a complex Frotelngemlaoh, which on the concentration duroh ultrafiltration, whereby la dynaalsoher Catfish In a «closed circuit In Va-Jniua is worked, and on the fractionation duroh decanting Proceed in an electroporethic convention is based.

Ss is well known, defl many of the characteristic ones Bjlgenso had a protein from its molecular structure and depended on the arrangement of the lsi NoIeIcQl present Eleeente. Both nerkaale are very sensitive and are for that Exposure to a great deal of friction from physical oils and ohas above Funds responsible.

Belsplelswele · can reduce the biological activity of Bnsynen, of Plastic proteins and protein hormones already exist by means of "ild procedures" which do not involve any of the

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lent weak acids, action of organic solvents (Ether * ethanol etc.), dissolution in the presence of high concentrations of certain salts (an exclusively physical or mechanical appearance) are further means of using whose molecular structure can be changed to a greater or lesser extent. This process of changing molecules are called denaturalization. The most striking effect of denaturalization is the decrease in solubility * with which a decrease in biological activity goes hand in hand. The coagulation of proteins through Warnings, for example from egg whites, is a well-known example for denaturalization. Other consequences of denaturalization are increases in the reactivity of amino acid chains and changes in optical rotation. This latter phenomenon is related to the fact that the enveloping of the Polypeptide chain gives the proteins an optical activity, whose sum is the aaymetrisohan carbon atoms of the amino acid components is equivalent to·

The isolation of a pure protein from a complex one Oemlsoh of protein components and non-proteinaceous components a biological tissue or fluid generally requires the use of a number of Procedures Every case has its specific difficulties, which depend on the properties of the protein to be isolated and depend on the nature of the auaganga material. Through the High instability of the green part of the proteins results Restrictions on the conditions that exist during isolation Can be applied to denaturalization too avoid «in the case of a biologically active protein "An enzymatic or hormonal activity as a measure of the changes" that occur during isolation can be taken.

1Q9811 / 2Q34 ^ßÄD

However, if there is no such biological activity, then it is often impossible to ensure that the isolated product has the same molecular properties are characteristic of the state of nature, i.e., of the state In the living cell or in the biological fluid. In order to largely increase the possibility of denaturalization reduce, certain general rules are observed in an expedient manner, which differ from the experience with known Deriving proteins: 1. Temperatures must be kept as low as possible, which is particularly important when organic solvents such as ethanol and acetone can be used. 2. Extreme pH values should be avoided. 3 «Only medium or high protein concentrations should be used used as most of the proteins in too much diluted state is relatively unstable. 4. Too vigorous stirring or peeling should be avoided, as the proteins on surfaces and interfaces more easily denaturalized will.

At present, the ObIlohen processes for separating the complex protein mixtures in an industrial and ha Ib industrial area Scale on the following methods:. 1. Precipitation of old neutral salts in high concentrations

(preferably ammonium sulfate).
2 »Fractionation by the technique of Cohn et al (ethanol) at low temperature) and the technique of Kakwlck and

Maohey (ether) «.

These techniques pose numerous difficulties, Also, according to a report by the Committee of Experts of the OMS on the employment of the human immunoglobulines * (Dr. Mund. Salud. Ser * Info Te «. 1966, J27, page 8) not .completely satisfactory as it changes in the plasmatic proteins and because they are also excessively expensive.

109811/2034

BATH ORIGINAL

A process has now been created with which it is possible to separate the proteins in a highly pure state using a closed circulatory system: (Always under Pyrex glass or a sterile plastic tube), a temperature of 4 ° C and an abiotic chamber will. This procedure is carried out as follows:

First, a protein mixture (plasma or human or animal serum, which is immune or not can) the inherent ions and mineral salts as are the ηloht-proteinic components with a low Molecular weight removed by means of dynamic ultrafiltration in vacuo and in a closed circuit. The above concentrate is then dissolved in a buffer solution, whose pH value corresponds to the isoelectronsohe point of the protein to be isolated. The protein ' mix in half the protein concentration of the original Raw material bubble · possessed.

The fractionation is then carried out with the aid of electrophoretic acid Convection carried out.

This is followed by the one from the previous procedure Isoelectric effluent obtained by means of dynamic ultrafiltration using a closed circuit is concentrated, with the ions, mineral salts and small molecules it contains, which are impurities represent, be removed. A sterile concentrate of pure protein (normal immunoglobulins or specific Iaaunoglobulln) received.,

After performing continuous fractionation electrophoretic convection is used with the non-isoelectric Downstream the same process In an an -

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their system for dynamic ultrafiltration in vacuum and In closed circuit and under electrophoretic Convection carried out.

Two sketches now follow for the implementation of what has been described Procedure for separating the immunoglobulin and albumin contained in one liter of human plasma are included »and also another sketch, which Details of Fraktlonlerungskapazltät and the Arbeltswelse a semi-industrial plant for the implementation of the new procedure.

109811/2034

BATH ORIGINAL

Sketch for the mode of operation in which (in a continuous operation) the proposed Protelnfractionationsystetn is used.

Expiry of the monthly work 4 periods of 144 hours (6 days χ 24 hours), which can be switched by rest breaks of 24 hours the filter, for sterilizations »cleaning and general Maintenance interrupted (the program corresponds to the opening time of the plasma or blood bank that supplies the system). Total working time is 576 hours

SKETCH

1. Fractionation
343 * 6 1 with 600 nl / et

Isoelectric Koopontnt ·:

990 * 932 1 old a »salary swlsohMi 2, $ and 4 kg ganae-Olobulln in Ä Iu

Non-isoelectric · component;

100.268 l, which contain dl · reetllohen plasma proteins. SI «has been concentrated to 20.053 l and on 401,072 I vsrdUnn t · "'

Fractionation old I 1 / st Non-risk component?

57 * 296 I, which the remaining heights Contain proteins.

AU3QAMQSPLASHA

Monthly amount of 345 * 6 1, You will be focused on 69 * 12 I. and diluted to 691.2 liters.

2. Fractionation! 6 1 with 600 ml / st

Isoelectric «component:

343 * 776 1, which between 8,6 and 15,000 kg of pure albumin contained in a wÄaarlger solution,

109Θ11 / 2034

BATH ORIGINAL

Sketch of the fractionation carried out by the proposed system. The sketch refers to the separation of the gamma globulin and the albumin in one liter of human plasma.

1,000 ml plasma (4.5-6.5 % proteins)

By dynamic ultrafiltration in a closed circuit and by means of vacuum up to 200 ml are found. Removal of 70-80 % of the originally present ions and mineral salts,

It gets old in buffer solution
a pH equal to the isoelectric point of gaeeaa globulin diluted to 2,000 al.
The ions and salt remain
at a concentration of 15
up to 20 % of the original
Concentration. Proteins 2.5 to

Fractionation by continued electrophoretic convection Flow: 600 ml / st tent 1.6 st Isoelectric component: 1 720 al (0.4 to 0.6 proteins)

Concentration up to * ü 56 al through dynamic ultrafiltration »removal of iron 70 to 80 £ ions. Dilution up to 1 120 ml, ions and acid 7 to 10 %.

Fractionation by electrophoresis Continued convection FluOt 1 l / st tent: 1.12 st, 96O ml (3 % protein approximately)

Concentration of the pure product by dynamic ultrafiltration and removal of ions by fractionation in 1.12 st. Between 25 and 35 g of pure albumin in aqueous solution are obtained obtain.

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In the attached drawing sketch is the procedure and the Technique of the present invention explained in more detail.

In the drawing, 1 denotes a piston into which the starting plasma is introduced and which served as a supply. At 2, a container is shown which serves as a regulating container. 3 designates the first battery for dynamic and vacuum ul "traftItratlon. 4 is a peristaltic pump, which is provided at the outlet of the battery. Next follows the calibrated ampoule 5 for concentrated and deionized liquids o In a second flask 7 the first solution to be fractionated follows produced, which consists of half a dilution of the original plasma in a special buffer solution »The solution contained in 7 is fractionated ^{by means of the device for electrophoretic convection 8 and 8 C.}

The isoelectric outflows and the non-isoelectric outflows are treated in two independent circuits that work simultaneously. In the first cycle the isoelectric The effluents are initially collected in the flask 10 and passed through the second ultrafiltration battery 11, the latter. exit leads to the peristaltic pump 12. This is connected to the piston 13, in which there is a pure solution of immunoglobulin collects.

The non-isoelectric effluents withdrawn through 9 are similarly collected in a flask 14. The concentration of proteins in this solution must be between 16 and 20 % . This solution goes to a regulating piston 15 and from there to the ultrafiltration battery (which in this case has a smaller module than the previous one). The solution then runs through the peristaltic Puape 17 and then to the Ampoule 18, where the deionized

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BATH

Concentrates are collected. Dilute runs out of the container 20 Buffer solution too, so that the solution to be fractionated to a quarter with respect to the non-isoelectric component is diluted. The electrophoretic device 21 fractions the non-isoelectric effluents that arise during the first fractionation ο the regulating piston 22, the fourth ultra-filtration battery 23, the pearl-shaped one Pump 24 and the collecting container 25 for albumin solution (which can be used directly in pharmaceutical preparations) make up the remaining parts of the systems.

It is pointed out »that in the described system the individual parts do not necessarily have to correspond to the description, but modifications are also possible as long as the essential characteristics are not changed.

The system has the following advantages:

1) The isolated proteins are obtained in high purity.

2) There are no denaturalizations.

3) High yields are obtained.

4) The cost of the system and the reagents are low.

5) It becomes practically all of the initial biological activity maintained.

10 ^ 811/2034

Claims (4)

Claims fly Process for the separation of highly pure proteins from complex protein shells, characterized in that it is in a closed circuit whose elements are made of Pyrex-Olas or consist of a sterilizable plastic tube, at a temperature of 4 ° c and in an ablotic Chamber is carried out, the process essentially from concentrating the protein smell and eliminating its ions, mineral salts and small molecules dynamic ultrafiltration in a vacuum in a closed Cycle exists. 2. The method according to claim 1, characterized in that immediately thereafter a dissolution of the concentrate in one Buffer takes place, the pH of which is equal to the isoelectric point of the protein to be isolated « 3 * Method according to one of the preceding claims, characterized characterized that the solution with the help of elektrophoretIsoher Convection is fractionated. 4. The method naoh one of the preceding claims, characterized characterized in that dl · concentration of the collected laoelectrlsohen Components whose ions and mineral salts have been removed, bit with the help of dynamic ultrafiltration Runs in a closed circuit. 5 »Method according to the preceding claims, characterized by the fact that dl · arises during ultrafiltration« UJaung under elderly conditions in the form of pure ·· protein giMMiSIt is »daa directly in pharmaceutical preparations can be used. - Il - 109811/2034 6 «Method according to one of the preceding claims, characterized characterized in that the fractionation by electrophoretic Convection resulting non-isoelectric Component at the same time the same process in another similar device for dynamic ultrafiltration and is subjected to electrophoretic convection, the product also being obtained in a purity which is suitable for incorporation into a pharmaceutical preparation. 10 * 811/2034 BATH fcl Blank page