DE2339111C3 - Process for the purification of proteins and other biospecific adsorbable substances - Google Patents

Description

The invention relates to a method for cleaning of proteins and other biospecifically adsorbable Substances by biospecific adsorption and by desorption.

On the one hand, the high costs of producing proteins, especially pure proteins, are relative low concentrate!;.:, of the protein in the biological. Material, but on the other hand in the elaborate multi-level Concentration and purification justified.

The protein is obtained according to several standard techniques, which are used in alternating succession in practically all processes. By mechanical, chemical, cnzymaiibcnem and / or tne / mixers digestion of the raw material, the clarification and concentration of the Rohextra ¹ 'is followed by the enrichment of proteins by precipitation with salts, acids, bases, solvents or by adsorption as well as by dialysis, ultrafiltration is carried out and dicfiltration.

This is followed by the actual purification of the protein, i. H. the separation of the protein sought from a large number of undesirable proteins. The following cleaning methods are known so far:

Fractional precipitation and adsorption, as well as elution and separation by molecular weight Gel permeation and diafiltration and such

Separation according to charge, isoelectric point and Ultrazer.trifugation.

As a rule, only incomplete cleaning can be achieved with these methods.

A few years ago the process of biospecific adsorption (affinity chromatography) was developed, which is based on the ability of many Protc: nc to recognize gcreisac rviuicKüic and to adsorb it strongly. The recognized molecules can be very different both in their chemical structure and in their molecular weight; so z. B. amino acids or proteins, ADP, nucleic acids and other molecules specifically recognized by the corresponding proteins

This strong adsorption now allows both 6s proteins and the substances they recognize to be purified by biospecific adsorption. The protein or the substance recognized by it is recognized a solid, chemically inert, not non-specifically adsorbing, water-wettable carrier of large size Surface bound; the substance bound to the carrier kovaient is said to be "Affinor" in the following to be named.

So far only granules (e.g. cellulose powder) or hydrophilic gels (e.g. Polysaccharides, polyarylates) are used.

Affinity chromatography is used to isolate the known substance or protein in such a way that its solution is passed over the effinor-substituted carrier, all undesired low- and high molecular weight by-products that pass through the carrier without delay are washed out and the biospecifically adsorbed product is then changed by changing the pH value, the salt concentration, Addition of substrate, innibitor (competitive displacement), effector, etc. is eluted.

The conditions to be used are often very harsh, and not only those that cause adsorption non-covalent bonds between the carrier-fixed Substance and the substance to be isolated, but also the tertiary and / or quaternary structure of the Protein-stabilizing bonds (also non-covalent bonds) can be cleaved. Since the Denaturation of proteins is an often slow process (high activation energy), the proportion is denatured Protein strongly dependent on the F. lurion time.

When transferring this method to the technical scale, the following difficulties arise:

The carriers previously used for biospecific adsorption are expensive.
Their chemical properties, but above all their physical properties, are not sufficient for technical use, because neither the shear forces that occur in batch operation due to stirring nor the pressures that occur in somewhat larger columns can be mastered.

In many cases, the protein is subject to the same conditions of desorption (both in batch, but especially in the column method) exposed for a relatively long time This means that the proportion of denatured Protein high.

The long residence times in the column or in the batch are understandably also for economic reasons Reasons disadvantageous.

The invention has for its object to eliminate the disadvantages of affinity and significantly decreasing the protein in the desorbed milieu in front of old ^v enveilzeit.

It has now been shown that this object can be achieved in a surprisingly simple manner if, in the cleaning process of the type mentioned, the adsorption on an ultrafiltration membrane is enoigt, on the product-side surface of which the biospecific adsorbing groups (affiners) are covalently bonded, and if the desorption of the biospecifically adsorbed product is carried out by reversing the flow of the desorbent (from the diaiysate to the product side).

An advantageous embodiment of the method according to the invention is that the Uiirafütrationsmembrane substituted with hydrophilic highly hydrated organic polymers to increase the surface area is and these polymers with the biospecific

adsorb ^ flu (Affinor) are substituted. When Substituents are preferably polysaccharides, polyvinylpyrenekk> n, polyacrylic acid derivatives (esters, -amid md copolymers ^ This allows the biospecific adsorption capacity (biosDecifically adsorbed Substance per unit area M; mbrane) of the membrane can be increased significantly.

With the method according to the invention, in addition to solving the problems mentioned, the biospecific adsorption - even the handling the diafiltration and the biospecific adsorption in one step, which also enables a considerable amount Simplification dsrstciit

An important step of the method according to the invention is the desorption by the task on the dialysate side of the desorbing medium on the membrane - possibly with a slight overpressure opposite the pressure on the raw product side - with simultaneous or subsequent removal of the desorbing Product due to the liquid circulating on the raw product side, which is much milder than that of the Desorption required medium can be.

The desorption method according to the invention is therefore much gentler than the desorption according to previous methods, because only in the thin layer the membrane surface the protein is exposed to the desorbent and very quickly into the product side circulating fluid enters. The otherwise three-dimensional distribution of the protein in the carrier, whether as a Affinor, or as an adsorbed product, is replaced by a quasi two-dimensional scale, the third being Dimension (perpendicular to the MeiTibran surface) rapid Allows desorption and short contact times with the desorbent

In the following, the implementation of the method according to the invention and the production of the membranes used for this purpose are explained in more detail with a few examples

Example 1 ^ ₀

A. Membrane, immobilized trypsin inhibitor

Regenerkrie Ceöulose-UItrafilter were activated in the Ultrafütraüonskanuner for 10 minutes with a solution of BrCN (25 mg / ml) at 20 bit 25 ^C C reacted with o-aminobutyric acid The activation of the COOH function was carried out with soluble carbodiment; Thereafter, msgeseizt was carried out with an excess solution of trypsin inhibitor (soybean wages). The membrane contained per m ²

B. HohlfaseTTBOälii! with biospecific activity versus guaniadeaminase and xnthinosidase

A hollow fiber module rail IGQ ml content equipped with CcHüloscTaEer ?! sows eissr muscular weight exclusion limit of SS® wmde, cne in A. bes ± rubbed, with activated ms! directly rest a solution of

pfänenyllguanin implemented (BR HAI Säsbesssick. |. Msd. Oism. t4

er! islt € $ is

of guanindeamine.'se and xanthine oxidase compared. The conditions for solvents. pH, concentration -'- • Mion and temperature corresponded to the conditions according to the cited reference.

The elution of guanine deaminase was carried out by adding 1.0 m molar guanine to the eluate after 5 Minutes of spontaneous incubation. Xanthine 'oxidase was with 1 m molar Hypoxantine - applied on the eluate side - eluted. The recovered enzyme activity was over 90%.

Example 2

3iospecific adsorption of ribonuclease on modified throat membranes

y-rays graft polymerized membranes according to the Literature (A. G. H off mann, G. Schmer, C. Harris, W.G. Kraft, Trans.Amer.Soc. Actif Int Org. 18 [1972], 10-16), were used in the filtration device at 20 ° Pr for 10-15 minutes with 10% Cyanogen bromide in 0.2 mNaiCOj activates the pH value was held at 15.0.

a) After a short time 10% solution

from

Wash out was with a 5 '- {4-AminophenyIphosphoyJPVp (cyL phosphate) in a Carbonate buffer solution of pH 9 was added and kept at 4 ° C. for 24 hours.

b) After washing with a 15% solution of ε-aminocaproic acid in a buffer solution of pH 9 offset After the reaction had ended (several hours) the carboxyl group was activated with water-soluble carbodimide (30 minutes) and with 5 '- {4-Aminophenyiphosphoryl) uridine-2' (3 ') - phosphate, the structural formula of which is shown below, implemented

H ₂ N-

0-P-OCH ₂ ~

ι ι y ° \

ö. V N

O O

55

K & ipsx vnsius that

^ fesfsfarsn doichführung. The asmh

"Sea S. S. Baker) bsi J O-

Bd VerwerßdiJ ^ the ajf this way manufactured membrane zor pure ^ mg of ribonuclease was the capacity of the usier a) obtained metnsrasie zymgr cnv ^ Membraai ^ dj ^ the tsater b) described membrane had iron et & ss straighter K & paznäi. however , showed stronger Msoxpusmasr RgxMmclease.

BeJspee! 3

sms

other

Graft polymerization with Hydroxyäthylmctacrylal - carried out analogously to the processes described in the literature - a product was obtained which was around showed little changed ultrafiltration properties. Here, too, it was activated by the method described in Example 2 and reacted further as under a).

When the membrane produced according to this example was used to purify ribonuclease, a binding capacity of 10 " ² g ribonuclease was found. Elution took place very quickly by adding 0.3 M acetic acid on the eluate side and rinsing the product with 0.05 M acetic acid.

Claims (2)

2339 11 i claims: 1. Process for the purification of proteins and other biospecific adsorbable substances by biospecific adsorption and by desorption, characterized in that the Adsorption on an ultrafiltration membrane takes place, on the product-side surface of which ^ ie biospecific adsorbing groups (affinors) are covalently bound, and that the desorption of the biospecific adsorbed product is carried out by reverse flow of the desorbent. 2. The method according to claim 1, characterized in that the ultrafiltration membrane for Increased surface area substituted with hydrophilic highly hydrated organic polymers and these polymers are substituted with the biospecific adsorbing groups (affinors) are.