RS66801B1 - Method for the simultaneous isolation and purification of transferrin and immunoglobulin g from serum - Google Patents

Description

Technical field

The present invention relates to a new procedure/method for the simultaneous isolation and purification of transferrin and immunoglobulin G from mammalian serum, whereby high purity proteins are obtained.

Technical problem

The technical problem solved by the subject application is the finding of a new procedure for isolating and purifying transferrin and immunoglobulin G from mammalian serum.

In more detail, the present application presents a new technical solution for a new procedure for the simultaneous isolation and purification of transferrin (Tf) and immunoglobulin G (IgG) from mammalian serum, whereby transferrin and immunoglobulin G proteins were obtained with a purity of 298 and 99%, respectively.

State of the art

Since the 1960s, the compound 7-ethoxyacridine-3,9-diamine 2-hydroxypropanoic acid (rivanol) has been used in serum protein fractionation methods. Over the decades, scientists have worked to find a method to obtain preparations of specific serum proteins in a simple and fast way, such as, for example, transferrin and immunoglobulins, preferably as pure as possible. Due to its ability to bind serum albumin, rivanol can potentially be used to isolate and purify other serum proteins, such as, for example, ceruloplasmin and haptoglobin.

Patent application CN1042716A describes a method for removing human serum albumin from human plasma, where the gradual addition of a rivanol solution precipitates human serum albumin (HSA) from a serum sample. The procedure was compared with the traditionally used cold ethanol precipitation method (Cohn's process), compared to which this procedure is faster, simpler and characterized by a higher amount of HSA removed from the serum (as much as 86.5%). Basically, this procedure is based on the same principle as the first step of the procedure from the subject application, and there any similarity ends because it does not describe further purification of transferrin and/or immunoglobulin G from the obtained sample, but only albumin, unlike the subject application.

European patent EP 2393834 V1 refers to the process of purifying a relatively impure transferrin solution using anionic and cationic chromatography, which includes two different chromatographic steps, whereby this method is significantly longer than the procedure described in the present application. Also, this method does not describe the isolation of immunoglobulin G, i.e. it does not produce igG, unlike the application in question.

Ana Penezic et.al.: A microscale protocol for the isolation of transferrin directly from serum. Clin Chim Acta.

2017 Aug;471:12-16 describes a method that successfully isolates transferrin from human serum using three precipitation steps and without the use of chromatography as a purification tool, the initial sample volume is 250 μL, and the duration of the entire procedure is only 90 min. The yield of this method is 58%, while the purity of the obtained transferrin is 97%. However, immunoglobulin G is not obtained with this procedure, and the method is only suitable for small sample volumes.

In the publication Sagan Z.: Application of rivanol for serum transferrin and immunoglobulin G determination, Clin. Chim. Acta. 1968 Aug; 21(2):225-30, described the isolation of immunoglobulin G and transferrin using rivanol (ethacridine lactate) as a precipitating agent. Protein separation is achieved by adding a 1.5% aqueous solution of rivanol to an equal volume of serum, followed by centrifugation. Immunological examination of the samples confirms that neither immunoglobulin G nor transferrin are found in the precipitate, while the supernatant does not contain albumin. However, in this work, unlike the present invention, these two proteins are not separated from each other, but only separated from other human serum proteins.

In the publication Al-Mashikhi et al.: Separation of immunoglobulin and Transferrin from Blood Serum and Plasma by Metal Chelate Interaction Chromatography, J Dairy Sci. 1988 JuI;71(7):1756-63, describes a procedure for separating a mixture of immunoglobulin G and transferrin from other proteins from cow serum, using metal-affinity chromatography. This procedure does not require sample preparation, but it also does not separate transferrin from immunoglobulin G, but only partially removes impurities in the form of human serum albumin.

In the state of the art, no method has been found that describes the simultaneous isolation of two separate protein preparations, immunoglobulin G and transferrin, by one procedure. Namely, in relation to the above-mentioned state of the art, the present invention describes a quick and simple procedure for the simultaneous isolation of immunoglobulin G and transferrin in separate fractions, whereby proteins of extremely high degree of purity are obtained, i.e. >98, i.e. 99%.

Also, additional advantages of this procedure are the semi-preparative purification scale and the flexibility in choosing the buffer system in the chromatographic step, where three different buffer systems were tested that showed reproducible results.

In relation to the methods described in the available patent and non-patent documentation, the present invention enables the simultaneous isolation and purification of immunoglobulin G with transferrin from a serum sample as a starting material, whereby isolated and completely separated proteins of high purity are obtained in different chromatographic fractions.

The essence of the invention

The invention provides a quick and simple procedure for simultaneous purification and isolation of transferrin and immunoglobulin G of high purity (>98, respectively >99%) in separate fractions from mammalian serum as starting material.

In more detail, the procedure includes the simultaneous isolation and purification of transferrin and immunoglobulin G carried out using two steps, the first step of precipitation and the second step of chromatographic separation, whereby the proteins obtained according to the procedure described here are over 98 and 99% pure, depending on the buffer system used.

The proteins obtained in this way can be further used directly, without additional purification.

The duration of the entire procedure described here is less than 80 min, and requires only two simple steps, one precipitation and the other chromatographic.

Transferrin and immunoglobulin G obtained by this procedure/method are in their physiological forms and are obtained in separated fractions.

Short description of the pictures

Figure 1 Isolate purity visualized by silver staining of SDS PAGE gel under reducing conditions for all three buffer systems. A-heavy chain of immunoglobulin G; B-light chain of immunoglobulin G; C - transferrin. Lanes 1,4 - final products, MES buffer; lanes 2, 5 - final products, BisTris buffer; lanes 3.6 - final products, phosphate buffer. The last lane contains molecular mass markers.

Figure 2 Representative chromatogram of separation of transferrin and immunoglobulin G using ÄKTA Avant 150 FPLC system, Resource Q column and MES buffer system.

Figure 3 Representative chromatogram of separation of transferrin and immunoglobulin G using ÄKTA Avant 150 FPLC system, Resource Q column and BisTris buffer system.

Figure 4. Representative chromatogram of separation of transferrin and immunoglobulin G using ÄKTA Avant 150 FPLC system, Resource Q column and phosphate buffer system.

Detailed description of the invention

Transferrin is an iron-binding glycoprotein with a mass of 76 kDa that is synthesized in hepatocytes. It is present in plasma, where its concentration is e.g. in the serum of healthy adults about 2.5 g/L, and in addition to the serum it is also present in the interstitial and cerebrospinal fluid and in the lymph.

Immunoglobulin G is a 150 kDa glycoprotein that belongs to the immunoglobulin protein family. This protein is one of the most abundant proteins in the serum, where its concentration in healthy adults is between 10 and 25 g/L. Immunoglobulin G has a function within the adaptive immune system and effector mechanisms of innate immunity.

A quick and simple procedure for the simultaneous isolation and purification of transferrin and immunoglobulin G of high purity >98, i.e. 99%, from serum as starting material is described. The procedure involves the use of one precipitation and one chromatographic (FPLC) step. The same results are obtained by using three different buffer systems during the chromatographic step, i.e. using MES, BisTris or phosphate buffer system. Proteins isolated and purified in this way are found in their physiological forms, i.e. this process of isolation and purification does not affect their structure and function.

The biggest challenge when isolating these two proteins individually and simultaneously is the presence of albumin, the most abundant serum protein. In numerous protocols for isolating one of the two mentioned proteins, albumin is the most common impurity in preparations of transferrin or immunoglobulin G precisely because of its presence in serum - it is difficult to completely remove it. Most protocols for the individual isolation of these two proteins, after removing most of the albumin, include additional chromatographic purification, as described in the present application.

In the subject application, albumin is removed from the serum using a rivanol solution. After processing the serum with rivanol solution, it is necessary to remove the rivanol remaining in the supernatant. This is achieved by applying filtration through activated carbon. This step is fast and efficient, but during it a part of the target proteins (transferrin and IgG) is lost, thus reducing the yield, but extremely high purity is achieved. During the chromatographic step, transferrin and IgG are further purified and separated from any residual impurities, resulting in the aforementioned high purity preparations.

Most commercially available preparations of transferrin and immunoglobulin G are of lower purity than the preparations obtained by the method described here, which is extremely important for use in fundamental and applied research, as well as for immunization of animals to obtain specific anti-Tf/anti-IgG antibodies. The same can be said for the largest number of preparations obtained by methods described in works or currently known patent literature. Impurities present in commercial preparations mainly come from albumin.

The novelty of the method described here lies precisely in the fact that two different high-purity serum proteins are simultaneously isolated and purified in one procedure.

In this way, both the consumption of the sample (serum) and the duration of the process are reduced, given that both proteins are isolated from the same serum sample at the same time. Also, by applying this separation and purification procedure, there is no need for additional subsequent purification of protein preparations.

In short, the present invention provides a new technical solution by which two separate protein preparations, transferrin and immunoglobulin G, of >98 and 99% purity are obtained from one sample of mammalian serum, using the same method, in a short time as products, which can be further used directly, without the need for additional purification.

This represents a novelty in relation to the methods described in the available state of the art in which protein preparations of approximately the same degree of purity are obtained from two different samples using two or more methods, with additional purification and greater time consumption.

The procedure includes a precipitation step, during which most of the albumin is precipitated, then an aliquot of the individual's serum is treated by slowly adding an aqueous solution of 7-ethoxyacridine-3,9-diamine 2-hydroxypropanoic acid, concentration in the range of 0.2 - 4% of the solution, rN~6 in a volume ratio of 0.1 - 2.

Mammalian serum includes human, bovine, and ovine serum, as well as serum derived from other mammalian animals. The chromatographic step involves the use of an FPLC system and a strong anionic ion exchange column, as well as a selected buffer system (MES, BisTris or phosphate) with a concentration between 20 and 75 mM. Within the chromatographic step, it is necessary to first equilibrate the ion exchange column, which is achieved by applying a selected buffer system with the addition of sodium chloride at a concentration of 75 to 200 mM, and then the same buffer system without added sodium chloride. After equilibration, the sample is applied to the column, then the column is washed, during which immunoglobulin G is obtained, and then elution, during which transferrin is obtained.

Materials:

7-ethoxyacridine-3,9-diamine 2-hydroxypropanoic acid (rivanol)

FPLC - Fast Protein Liquid Chromatography

MES – 2-morpholine-4-ylethanesulfonic acid

BisTris-2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

ÄKTA Avant 150 FPLC system

Resource Q, 6 ml - Strong anionic ion exchange column;

SDS PAG electrophoresis - sodium dodecyl sulfate polyacrylamide gel electrophoresis

Commercial putty for painting with silver

Example

The process of the present invention is illustrated by the following example, and includes the following steps:

1. The precipitation step during which most of the albumin precipitates, an aliquot of mammalian serum is treated by slowly adding (drop by drop) 0.2-4%, preferably 1.2% aqueous solution of 7-ethoxyacridine-3,9-diamine 2-hydroxypropanoic acid, pH~6 in a volume ratio of 1:1.

I. The precipitate obtained in this step is separated from the supernatant by centrifugation at 4500 rpm for 15 minutes.

II. The resulting precipitate is discarded, while the supernatant is filtered through activated charcoal to remove excess rivanol from the solution.

III. Then the pH value is adjusted to 6.2 with the clear supernatant.

2. The chromatographic step involves the use of an ÄKTA Avant 150 FPLC system and a strong anionic ion exchange column (Resource Q, 6 mL), as well as a selected buffer system, i.e. MES, BisTris or phosphate, concentrations between 20 and 75 mM, preferably 20 mM. Column equilibration takes place at a flow rate of 1.5 mL/min, and is achieved by using a buffer containing sodium chloride with a final concentration in the range of 25 mM to 200 mM, preferably 125 mM, and preferably 175 mM (elution buffer, 3 column volumes), and then using the same buffer without added salt (washing buffer, 3 column volumes).

I. After equilibration of the column, the sample application step follows, in which 1 mL of the prepared sample is applied to the column at a flow rate of 0.75 mL/min. Then follows a phase of washing the column with washing buffer at a flow rate of 1.5 mL/min (2 column volumes), during which immunoglobulin G is obtained.

II. The final phase of the chromatographic procedure is the elution phase, which involves the gradual introduction of elution buffer into the system in steps of 25, 50, 75 and 100% elution buffer (2 column volumes per step), at a flow rate of 1.5 mL/min. In this phase, transferrin is obtained.

� The transferrin obtained in this way is for use as a molecular marker or standard in qualitative and quantitative analyses, as an addition to certain media for growing cell cultures, e.g. in the cultivation of embryonic stem cells and in cultures where "serum-free" conditions are necessary, and for immunization of animals in order to obtain anti-transferrin antibodies, i.e. for detection during histochemistry, cytochemistry and Western blot analysis and for immunoaffinity chromatography.

Immunoglobulin G obtained in this way is for use as a standard in qualitative and quantitative analyses, for immunization of animals to obtain anti-IgG antibodies, i.e. for detection during histochemistry, cytochemistry and Western blot analysis and for immunoaffinity chromatography, as a starting sample for isolating specific monoclonal IgG antibodies.

In addition, the transferrin obtained by the described process is for use in clinical research, e.g. transferrin can serve as a "deliverer" of anti-cancer therapy to the target cells, with a negative finding for the presence of human viruses and mycotoxins or with their removal and a subsequent negative finding.

Claims (5)

Patent claims 1. A method for the simultaneous isolation and purification of transferrin and immunoglobulin G from mammalian serum, indicated by the fact that it includes: I. treatment of the serum with an aqueous solution of 7-ethoxyacridine-3,9-diamine 2-hydroxypropanoic acid (rivanol), whereby the precipitate obtained is separated from the supernatant by centrifugation and discarded, and the supernatant is filtered through activated charcoal; II. applying the resulting supernatant to the FPLC system and a pre-equilibrated strong anion ion exchange column, where by washing the column with a buffer selected from the group consisting of phosphate, MES or BisTris buffer, immunoglobulin G is obtained, and by elution with the gradual introduction of a buffer selected from the group consisting of phosphate, MES or BisTris buffer with the addition of sodium chloride, transferrin is obtained. 2. The method according to claim 1, characterized in that the duration is shorter than 80 minutes. 3. The method according to claims 1 and 2, characterized in that the sample of mammalian serum is selected from human, bovine, and sheep serum, as well as serum originating from other mammalian animals. 4. The method according to claims 1 to 3 characterized by the fact that transferrin and immunoglobulin G are obtained with a purity of >98, that is, 99%, whereby both transferrin and immunoglobulin G are obtained in their physiological forms. 5. The method according to claims 1-4, characterized in that transferrin and immunoglobulin G can be used directly without additional purification, namely transferrin as a molecular marker or standard in qualitative and quantitative analyses, as an addition to certain mediums for the cultivation of cell cultures, for the immunization of animals in order to obtain anti-transferrin antibodies, for clinical research with a negative finding for the presence of human viruses and mycotoxins or with their removal and subsequent negative results; immunoglobulin G molecule as a standard in qualitative and quantitative analyses, for the immunization of animals in order to obtain anti-IgG antibodies, as an initial sample for the isolation of specific monoclonal IgG antibodies.