CA1064425A - Process for obtaining protein conjugates - Google Patents

Abstract

PROCESS FOR OBTAINING PROTEIN CONJUGATES
Abstract of the disclosure:
A process for the purification of protein conjugation differ from the organic compounds used for the conjugation in their electrophoretical properties, by subjecting the crude conjugate to a preparative electrophoresis method in order to separate it from undesired by-products and unreacted con-jugation components.

Description

The pre~ent invention relates to a process for obtalning pure protein or protein mixture3 ll~ked by covalent bonds to organic compounds and called proteln "con~ugate~".
Since the protein conjugate to be obtained differs from un-desired con~ugation product~ and from the organic compounds u~ed ~or conjugation in its electrophoretlcal properties, the proce~s ~or obtaining the de~ired compositions i~ based on a preparative electrophoresis of the abo~e mixture, carried out after the con~ugation reaction~ and o~ a sub~equent separation of the zone contalning the desired compo~ition~ from those zones containing undesired protein con~ugates and free organic compound~.
Thi~ i~ventlon e~pecially relates to a process for the purification of proteln-dye~tuff conjugates, preferably of antibody-containing immune sera or protein fraction~ that have been reacted by con~ugation with dyes, especially with flu-orescent dyes, for example fluore~cein isothiocyanate, by means of preparative electrophoresis.
Thi8 inventlon further relates to protein conjugates that have been purified according to this process and to their use in the immunological diagnosis.
Due to the el~ctrophoretical properties of the starting materisl and o~ the reaction products, the process of the in-~ention permlt~ the ~epar~tion of the pure protein con~ugate having a certain degree of reaction from unreacted protein and ~rom the organic molecule used for con~ugation, provided that the~e three component~ have a different electrophoretic mobillty.

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Protein-dyestu~f con~ugates, especially lmmune sera con-tainlng antibodies and ~on3ugated with fluorescent dyes, are particularly important in the so-called immunofluorescence technlque, as will be shown in the ~ollowing without limiting the invention to th~sa examples.
The immu~ofluorescence technlquet reported by A.H. Coons in 1942, permlts a comb~ned in~e~tigation of serologic-immuno-logic specificity and of the morphological characteristics of ; the material to be analyzed. This method i~ based on the so-called ~ntigen-antibody reaction, in which one of the two lmmunologic compon~nts - generally, the antibody - i8 con-~ugated with a fluorescent dye~tu~f. The anti~gen-antibody roactlon ~ompound i~ made vlsible in the microscopic preparation by provoklng the visible fluorescence by means of rays in the ultravlolet range.
The immunofluore~cence technique has acqulred great signi~icance in the diagno8is for identlfying linked and free antigen~ and antibodie~
According to the s~te of the art, fractions with a high concentratlon of antibodie~ are reacted as so-called immune sera with fluorescent dyes, preferably with fluorescein iso-thlocyanate. The reaction product is then predominantly purlfied by gel flltratlon and by ion exchange chromatography - on dlethyl-aminoethyl (DEAE) cellulose, whereby the fluorescein-conJugated immune globulins are i~olated.
Tho~e know~ methods have the drawback that the separation of the dyestuff, which is not linked by a covalent bond, from the fluorescein-con~ugated protein composition by means of gel 29 ~lltratlon or dlaly~is 1~ incomplete; in particular, a dyestuff ': , . .

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Compositions containing such free dyes lead to unspecific fluorescence Coloratlons in the~microscopic preparation and thus to false results.
The widely used prior art process for obtaining con~ugated protein fraction containing antibodies by means of ion exchange chromatography on DEAE cellulose generally provides only the antlbody fractlon belonging to the IgG class. Under the conditions applied for elution and separation, the an~i-bod1es of th~ IgM and IgA cla~ses are separated only unsa~is-factorily or are not separated at all, ~o that the preparation obtained partly shows a substantial 108~ of the desired specific antibodies. When modifying the elution conditions suitable for the recovery of fluore~cein-con~ugated IgM and IgA anti-bodios, so-called "overlabeled" ~-globulins as well a~ baso-phllic proteins are also eluted. Owing to their strongly acid nature, these "overlabeled" protein fraction~ lead, in the immunologlc reaction, to an un~pecific linkage to a number of basic proteins in the matorial to be examlned and thus again to a misinterprotation of the ~luorescence results.
The proces~ of this inventlon avoids tho~e drawbacks. m e eloctrophoretic purificatlon of dyestuff-con~ugated protein preparatlons, preferably fluoresceln-con~ugated proteln pre-paratlons, carrled out preferably ln an inert carrler material 29 aiter the con~ugation reaction, offers the following advantages:
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,, ' '... , .... ''. , ,., ` . ,. ,;. .,~ ~ ,, 1. The process permit~ an optimum rec~very of "acGurate" zone~
containing antibodie~. Undesired protein fractions that are, for example, free of ant$bodies can eas~ly be eliminated together with the separation of the de~ired fraction.
The yield of the dye~tu~f-conJugated protein preparations containing antlbodies i~ optimum. In an exRmplified case of a fluorescein-labeled ~-globulin fraction, 70 to 90 % of the amount used are reco~ered.

2. The con~ugatlon reaction of proteins with acidic organic molecules reduce~ the equipstential point~ in all the proteins reacted wlth the organic molecules, 80 that under the electro-phoresis condition~, the reaction products migrate toward the anode at a higher speed than non-con~ugated proteins.
There i~ no difficulty in eliminating proteins which have become too acidic owing to an intensive reaction with the molecule used for conJugation, since these migrate toward the anode at a higher speed. This ~inding iq especially significant with the proteins that are con~ugated with fluorescein isothiocyanate a_ the acidic fluorescent dyestuff (qulnoid sy tem with a ~ree carboxylic group).

3. The proces~ of the lnvention ensures the optimum separation of the acidic organic molecule, which is not linked (or llnked by ad~orption) and which, if present in the fluores-celn-con~ugated compositions, lead~ to undesired, unspecific fluorescence colorations. The reason for thiq easy elimin-ation of thi~ dyestu~f portion is that the acidic molecule itself has a ~trong tendency to migrate toward the anode.
The electric potential established under the conditions of ; 29 the preparatlve zone electrophore~is cau~es the molecules .
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which are relatively loosely bound to protein to be split of~
and enhànces the quality of the protein con~ugate~ recovered by electrophoresi~. In the case of protein~ conjugated with fluorescent dyos, the stabllity of ~uch preparation~, when ~tore~ in an aqueou~ ~olution, i8 lncreased in comparl~on with those obtained according to the state of the art.

4. When antibodie3 are used as proteins for the con~ugation, the process of the inventlon allows all the dyestuff-con-~ugated antibody classe~ to be obtained, for example IgA, IgG, IgM. This is of importance especially for the use of antibodies obtained from sera, for example of man, goat, sheep and hor~e, in which-besides the antibodies of the IgG class-al~o antibodies of other immunoglobulin classes account for a relatively hlgh percentage of the tdal anti-body amount. This factor 18 also of a substantial economic interest since antlbody-containlng sera are available to only a limited extent.
- U~ed for the purificatlon of antibodie~ con~ugated with fluorescent dyes, the proce6s of the invention brings about ~, 20 an increa~e in the relationship between specific antibodies (Ab) and total protein (P), the so-called Ab/P quotient, which again intonsifies the desired specific fluore~cence . .
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5. Tho proce~s, which ¢an be operated without using expensive chromatographical ad~uvents, results in a substantial cost reduction for the manufacture of pure protein con~ugate~.
~; The inert carrier material used may be employed ~everal ^ times.
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~ HOE 74/B 011 The preferable electrophoretic method used is the carrier electrophore~is, in particular the electrophoresis u~ing the inert carrier material in a horizontal container. When, for example, polyvlnyl chloride granules are used as the carrier material, it i8 possible after the ~eparatlon of the fluores-celn-con~ugated proteln fractions containing the antibodies to cut out the desired preparation in accordance with the visible bands and then elute it from the carrier by means of slmple solvents.
When the con~ugate i8 uncolored, the positions of protein, reaction component and proteln con~ugate can be established in-directly, for example by means of a paper dabblng which is then colored in ~nown manner.
In additlon to polyvinyl chlorlde, for example polyacryl-amldo, cellulo~e, starch, glass beads, sand and the like are frequently used as electrophoretic carrler materials.
Electrophoresls equlpment used accordlng to the lnvention also include vertical arrangements uslng carrier material or not. Very good puriflcatlon effects are also obtained using contlnuously operatlng equipment.
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The process of thi~ invention ~eparate~ antibody preparations of any origin, whlch have been linked by covalent bond to dyes :`
o~ acidic nature, especially to fluorescein lsothioc~snato.
It is, however, also possible to react non fluorescent ~ 25 dye~ wlth proteln~ and to separate the proteins colored by the ; conJugation reaction accordlng to the proces~ of the invention, provlded the dyes u~ed 8~0W different electrophoretlc migration properties.
29 In~tead of antibodies, other proteins o~ animal, vegetable . ~ .
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. . . : . , . : . , and microbial origln may also be used, if they exhibit a~ con-~ugates an electrophoretical property that differs from that of the starting protein and of t~e organic molecule used for the conJugation. Organic molecules preferably used for the con~ugation are compoundæ having their equ~potential points within the acld pH-range.
The essential feature of this invention reside~ in applying preparative electrophoretic methods for the manufacture o~
pure protein con~ugates, especlally of gamma-globulin fractions reacted with fluorescent dyes. The reaction of ~-globulin fractlons with eonventional fluorescent dyes is not expected to yield an electrophoretically homogeneous product, since the structure~ of the immunoglobulin classes used for the reaction are known to be diiferent and rather have a different number of amino groups available for the reaction. Although electro-phoretlcal methods are known to provide relatively uniformly mlgratlng zones of unroacted ~-globulina, the uniform migration of immunoglobulins reacted with dyes and belonging to different classes has to be regarded as a surprise. Moreover, the dyes linked to protein molecule~ by adsorptlon could not be expected to be ~eparated from the protein in an electric field. Finally, owing to the known intense linkage of fluorescent dyes to ion exchange material aa used in the state of the art, it could not be foreseen that the free dyestuff used in an electrophoretic method? especially together with polyvinyl chloride as a carrier material, migrates over a particularly long di3tance as com-pared to that of the protein con~ugates.
The known proce~ of eleGtrophoresls yleld~ a product of 29 aub~tantially hlgher purity than chromatographlcal methods do.

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HOE 74!B 011 The product obtained exhibits, as a surpri~ing technlcal effect, a higher ~peciflclty when used as an immunological reagent than the product obtalned accordlng to the state of the art.
The ~ollowlng Example~ illustrate the invention.
E X A M P L E 1:
A 3 % antibody-containing ~erum protein ~olution (goat) was reacted ln known manner with fluore~cein~ i~othiocyanate in an alkallne medium. In a horizontal electrophoresl~ equipment (size: 65.0 cm ln length, 76.0 cm in width, and 1.2 cm in helght), which contalned 3.6 l of PVC powder (Geon X 427, trade ;~ mark of MeQ~r~. Serva, Heidolberg, W.aermany), the con~ugated antiserum wa~ soparated at pH 8.1, at a field potential of 6 V
per cm, and at an amperage of 0.6 A, over a period of 15 hours.
The ~-globulin fraction containing the antlbodies, whlch generally remains unmoved at the application point or even . ,, mlgrates toward the cathode unle~s conJugated to fluore~cein :i'! i~othiocyanate, migrated clearly toward the anode. The light yellow to greeniJh coloration Or the ~-globulin fractlon con-talnlng the sntlbodle~ stood clearly out against the rest Or prot~ln fractlon~ and free dye~tuff portlon~ wlth their dark yellow to reddl~h brown color. The deslred ~-globulln fractlon containing the antibodles and havlng the optlmum F/P quotlent -which can also be determined bandwi~e l~mediately after separa-tion - was cut out as a PVC zone and washed clear of the i 25 carrier material by mean~ of a 0.9 X sodium chloride solution.
The eluate wa~ concentrated to about 1 g % of proteln by ultra-flltratlon.
Thls last operation was followed by the final standardization 29 and quallty control of the product.
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: ~,: : , 644~5 From 3~6 g o~ a fluorescein-con~ugated protein solutlon containing antlbodies (120 ml of antiserum), 0.5 g of a fluore~-celn-conJugated ~-globulin ~raction containing antibodies was obtained.
When ~erum proteins of rabbits containing antibodies were u~ed in an amou~t of 8 g of a fluore~cein-con~ugated antiserum containing antibodie~ (120 ml), 1.0 g of a fluore~cein-con-~ugated ~-globulin ~ractlon containing antibodies was obtained.
Analogou~ yields were obtained by recovering antibodies from human Qera. Considering that the proportion of the ~-globulin fraction in an anti~erum accounts for about 15 to 18 %, these yield~, calculated on thi~ ~-globulin fraction, c~n be regarded a~ optimum.
E X A M P L E 2:
A ~ % human tr~n~ferri~ solution was reacted in known manner with fluorescein isothiocyanate in an alkaline medium.
In a horizontal electrophoresis equipment (size: 65.0 cm long, 76~0 cm wide9 and 1.2 cm high) containing 3.6 l of a PVC powder (Geon X 427, trade mark o~ Messrs. Serva, Heidelberg, W.Germany), a con~ugated protein was ~eparated at pH 8.1, at a field potential o~ 6 V per cm and at an amperage of 0.6 A, over a perlod of 15 hourQ. The transferrin con~ugate clearly migrated toward the ancde. The yellow-brown coloration of the trans-ferrin-containing zone stood clearly out again~t the different colors of the ~ree dyestuff portions which had migrated still further toward the anode. The transferrin con~ugate having the optimum F/P quotient - which can be determined bandwise immadlately after separation - was cut out a~ a PVC zone and 29 wa~hed clear of the carri~r material by means of a 0.9 ~ sodium . ' 0; .
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10644~:5 chloride ~olution. The eluate was concentrated to about 1 g %
of protein by ultra-filtration.
This operation W&~ followed by the final standardization and qual1ty control of the product.
From 3.6 g of human transferrin, about 3.0 g of the cor-re~ponding ~luore~cein-con~ugated protein were obtained, ..

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Claims (11)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation and purification of a protein conjugate in which the protein is linked to an organic compound which differs in its electrophoretical properties from the resultant crude protein conjugate and the crude conjugate containing by-products and unreacted conjugation components is subjected to preparative electrophoresis to separate the protein conjugate from the by-products and unreacted conjugation components.

2. A protein conjugate, whenever obtained according to a process as claimed in claim 1 or by an obvious chemical equi-valent thereof.

3. A process as claimed in claim 1 in which the protein is an antibody-containing gamma-globulin fraction and the organic compound is a dye.

4. A protein conjugate, whenever obtained according to a process as claimed in claim 3 or by an obvious chemical equi-valent thereof.

5. A process as claimed in claim 1 in which the protein is an antibody-containing gamma-globulin fraction and the organic compound is a fluorescent dye.

6. A protein conjugate, whenever obtained according to a process as claimed in claim 5 or by an obvious chemical equi-valent thereof.

7. A process as claimed in claim 1 in which an inert carrier material is used in the electrophoresis.

8. A protein conjugate, whenever obtained according to a process as claimed in claim 7 or by an obvious chemical equivalent thereof.

9. A process as claimed in claim 1 in which the organic compound is a fluorescein-isothiocyanate.

10. A process as claimed in claim 9 in which the protein is an antibody-containing gamma-globulin fraction.

11. A protein conjugate, whenever obtained according to a process as claimed in claim 9 or claim 10 or by an obvious chemical equivalent thereof.