The present invention relates to novel methods for separating hemagglutinin (HA) antigens, comprising the steps of applying a reduced and derivatized antigen preparation comprising solubilized HA antigens and a detergent in a pH controlled solution, on a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) column; and eluting the HA antigens from the column with an ion pairing agent in an organic mobile phase. The invention further relates to quantifying methods using the methods for separating the antigens with the further step of measuring the peak area of the eluted antigen in a chromatogram resulting from the elution step.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention relates to methods for separating hemagglutinin (HA) antigens, comprising the steps of applying a reduced and derivatized antigen preparation comprising solubilized HA antigens and a detergent in a pH controlled solution, on a Reversed Phase High Performance Liquid Chromatography (RP-HPLC) column; and eluting the HA antigens from the column with an ion pairing agent in an organic mobile phase. One preferred embodiment of the present invention relates to a method according to the invention, wherein said elution is performed at a temperature between about 25.degree. C. and about 70.degree. C., preferably between about 40.degree. C. and about 70.degree. C., and more preferably between about 50.degree. C. and about 70.degree. C., and most preferably between about 60.degree. C. and 70.degree. C. In another preferred embodiment, the method comprises a step wherein the antigens are cleaved by a protease, such as trypsin.

The invention also relates to methods for quantifying the HA titer of an HA antigen preparation, said method comprising the method of separating hemagglutinin (HA) antigens according to the invention, with the further step of measuring the peak area of the eluted antigen in a chromatogram resulting from the elution step.

DETAILED DESCRIPTION

Here, a novel separation and quantification assay for the determination of hemagglutinin (HA) concentration by Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) is disclosed. The problem with the RP-HPLC methods that have been described in the art for separation of the antigens from influenza virus was that the separation was not optimal, with poor resolution of the protein peaks of interest, and that the recovery was low and not quantitative. The inventors of the present invention have now solved many of these problems by using a certain RP-HPLC assay in a set-up in which the antigen is reduced in the presence of a detergent, after which an inert derivatization of the antigen preparation is performed thereby protecting the sulfhydryl groups on the antigen. Preferably, for this step, an alkylating agent is added to render a reduced and alkylated antigen preparation and wherein the antigen is present in a pH controlled solution. It was found that by increasing the temperature during elution of the antigens from the RP-HPLC column, the recovery and the reproducibility of the assay was increased. The assays known from the art were performed at room temperature.

The inventors of the present invention have also found that it is preferred to select a column material that is suitable to be used at higher temperatures of up to about 70.degree. C. Preferred column material is therefore polymer-based, which generally can be used in these high temperature ranges. It is also preferred to keep the solution in which the antigen is dissolved pH controlled, preferably at neutral pH values. Preferably, values between about 5 and about 9 are used, more preferably values between about 6 and about 8 are used, while it is most preferred to use pH values between about 7 and about 8. Methods for buffering solutions are well known in the art and are herein not further elaborated on.

The virus preparation can be brought on the column, eluted from the column and the quantities of the antigens can be calculated from the specific peak areas all in a single day. It is thus a fast and robust method. Moreover, the methods clearly show that the process is accurate (as found in comparison to the SRID assay) and reproducible. The invention relates thus to a fast and accurate means for determining the HA concentration in different kinds of samples within the manufacturing process of influenza vaccines, thereby overcoming most of the problems associated with the methods known in the art.

In the disclosed assay, the quantification of HA is based on the peak area of HA1, which is well separated from the other vaccine components. The applicability of the present invention is demonstrated for different influenza A subtypes, including H1N1, H3N2, H5N3, and H7N7, strongly suggesting that the assay can be broadly applied for different hemagglutinin antigens. The Neuraminidase (NA) component of the strains is not limiting the broad applicability of the invention, as it relates to the separation of the HA component. It is assumed that the invention will also be applicable for influenza B subtypes, as well as for other viruses comprising hemagglutinin antigens that behave in a similar manner on HPLC columns.

The present invention relates to a novel method for separating hemagglutinin (HA) antigens, said method comprising the steps of applying a reduced and derivatized antigen preparation comprising solubilized HA antigens and a detergent in a pH controlled solution, on a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) column; and eluting the HA antigens from the column with an ion pairing agent in an organic mobile phase.

In one embodiment of the present invention, Influenza virus particles obtained from an upstream process of either egg-derived material or virus material from cell culture are first solubilized by the addition of a detergent, preferably a zwitterionic agent, more preferably Zwittergent, to a concentration of for example about 1% (w/v), but this is not critical to the invention. Subsequently, samples are treated with a protease such as trypsin (typically present on beads) to cleave all HA molecules into the subunits HA1 and HA2, which are only kept together by a single disulfide bridge upon this treatment.

As trypsin is hindered by the presence of high concentrations of SDS, SDS is preferably not used when the trypsin step is added to the method. If SDS is used, the concentration should be low enough not to inhibit the protease. Thus, it is preferred to use a detergent that does not inhibit the activity of the (possible) additional protease.

The disulfide bridge is then broken by addition of a reducing agent, preferably dithiothreitol (DTT) to a concentration of for example 25 mM, although other concentrations may also be used, and reduction takes place for about 10 minutes at about 90.degree. C. As in some cases the samples acidify to pH values of 4.0 and become slightly milky and turbid, it is preferred to perform the reduction under buffered (relatively neutral pH) conditions, such as in 150 mM Tris-HCl, pH 8.0. To prevent re-association and/or complex formation of HA1 with HA2 and other proteins, it is highly preferred to have the sulfhydryl groups of all proteins protected, for instance by the addition of an alkylating agent, such as iodoacetamide (IAA) or iodoacetic acid. Any inert derivatization of the --SH groups may be applied, such that no active groups remain. For example, any suitable sulfhydryl alkylating agent known in the art may be used. Examples of other suitable alkylating agents are N-ethylmaleimide; dithiobis(2-nitro)benzoic acid; nitrogen mustards, such as chlorambucil and cyclophosphamide; cisplatin; nitrosoureas, such as carmustine, lomustine and semustine; alkylsulfonates, such as busulfan; ethyleneimines, such as thiotepa; and triazines, such as dacarbazine. The person skilled in the art is aware of what compounds may be used to have the sulfhydryl groups protected and what compounds may be used to derivatize the antigen preparation, such that no active groups remain.

As disclosed herein, alkylation with IAA is usually performed at 37.degree. C. in the dark for about 45 minutes, but other conditions (T, time) will work as well. This step is then preferably followed by the step of adding an alkylation-inhibiting agent, for instance through re-addition of DTT to neutralize all remaining IAA molecules, upon which the samples are ready for RP-HPLC analysis. The HPLC analysis may be performed by using a POROS R1/10 column (Applied Biosystems), but other comparable columns would work as well. Usually, an acetonitrile gradient from 20 to 35% is applied at a column temperature that rnay be as high as 70.degree. C., but preferably around 65.degree. C., as the column cannot withstand temperatures that are much higher. In this high temperature range, the HA1 peak is generally highest for this column. When the column material allows higher temperatures, the elution temperature may also be increased.

The column of choice is usually selected for its performance at high temperatures. Although silica-based columns such as C4 or C8 can be used, polymer-based columns, such as the POROS R1/10 column, are preferred as they can still perform well at temperatures as high as 70.degree. C. As outlined herein, higher temperatures ensure a better recovery from the column, and thus in a better quantitative method.

In the course of the experiments, it was found that prolonged storage of the reduced/alkylated samples at 4.degree. C. may result in a deformation of the HA1 peak in the RP-HPLC graphs, which would influence the accuracy of peak measurements and thus on the accuracy of the method. Although it does not count for crude samples, present in medium, this would limit the storage/shipment possibilities of the treated (reduced/alkylated) samples and thus on the overall usefulness of the method. This problem of deformation of the peak was solved by adding the reducing agent (exemplified by DTT) also after the alkylation step, thereby decreasing the harmful effects of the alkylating agent. So, the additional step of adding a reducing agent after alkylation, is highly preferred.

It was also found that certain concentrations of DTT increased the peak area of HA1 when the samples were analyzed immediately. In general, it is preferred to use concentrations of the reducing agent that are higher than about 4.4 mM, more preferably at least about 11 mM, and most preferably about 22 to about 25 mM.

Generally, when the method of the present invention is carried out as a routine, using the same column for different runs, carry-over from HA from one run to another occurs (see for an example Van der Zee et al. 1983). To reduce the effect of carry-over, a wash step of the column with a detergent, such as 1% SDS or 1% Zwittergent is highly recommended between different runs on the same column, to remove all residual HA from the column material.

Hemagglutinin (HA) antigens are well known in the art. Although the method of the present invention has been demonstrated to work well for hemagglutinin antigens from influenza, it is likely that the method can also be applied for other hemagglutinin antigens derived from other viruses, such as measles virus. Thus, the present invention relates to a method for separating HA, wherein said HA is of an influenza virus or a measles virus. Preferably, said influenza virus is an influenza A virus or an influenza B virus. Also preferred are methods according to the invention wherein said HA is of an influenza A virus strain comprising an H1, H3, H5, or H7 hemagglutinin. For an accurate calculation of the HA concentration, it is preferred to have the HA0 mature form of influenza substantially separated into the subunits HA1 and HA2, as HA1 is generally the component which can be easily distinguished in chromatograms and of which the peak area can easily be assessed.

The reduction of the antigen is preferably pH controlled, i.e., buffered to a suitable pH. Typically, as described herein, a pH of about 8.0 was applied. However, other suitable (relatively neutral) pH values may be used, such as pH 7. It is important to note that the antigen precipitates in solution at pH values that are too low, for instance at a pH value of 4, or even lower. pH values between about 5 and about 9 may typically be applied, while more preferably values between about 6 and about 8 are applied, since at pH 6 the hemagglutinin antigen unfolds during the infection process under natural conditions. It is most preferred to use a pH value between about 7 and about 8. The person skilled in the art will be capable in finding the correct pH value with which the antigens are still acceptably separated, while it is also readily visible when a pH value is too low as the antigen precipitates at such values. As stated intra, it is well within the skill of the skilled person to adjust pH values and to buffer solutions. Typically, as used herein, a solution is buffered with Tris/HCl, but this is not critical to the invention.

Reduction of the antigen preparation is preferably performed using a reducing agent such as dithiothreitol (DTT). To prevent the antigens from re-association or from complex formation, it is highly preferred to have the sulfhydryl groups protected. This is performed through inert derivatization. Derivatization is preferably performed using an alkylating agent. Alkylation is preferably performed by using IAA. Especially when methods are performed in high throughput setting in which the antigen preparations may be left a prolonged period of time before application on the column, it is preferred to keep the antigens dissociated from each other, and thus to have no active groups present on the proteins. Alkylation may also be used for regular settings, or short-term methods.

The art discloses the use of an RP-HPLC method to separate influenza virus components (Phelan and Cohen. 1983). In this, viral proteins were solubilized and denatured in guanidine-HCl, and reduced by incubation with DTT for several hours at room temperature, wherein the pH was not controlled. Subsequently, analysis was performed by RP-HPLC at room temperature on a silica-based (C8) Aquapore column, applying a linear gradient of 0.05% TFA in water to 0.05% TFA in acetonitrile. However, the separation of the various virus components was far from optimal, whereas the recovery was low and not quantitative, presumably due to aggregation of the virus components and/or nonspecific adsorption to the HPLC system/column. Alkylation, or any other inert derivatization of the preparation was not applied, which may have resulted in re-association of components and unwanted complexes may have been formed. In addition, in this HPLC assay HA2 could not be detected, presumably because it had been trapped on the column matrix due to its strong hydrophobic nature.

Although the RP-HPLC method is generally quick, the teaching of the art was that it was not suitable for accurate measurements of antigenic determinants, such as HA of influenza. Guanidine-HCl, as used in the art, is a chaotropic agent that seems too harsh for the antigens and may even destroy some of the antigens of interest. Thus, such chaotropic agents are preferably not used for the methods of the present invention. Suitable detergents that are typically applied for the present invention are for instance SDS and several zwitterionic detergents. Examples of zwitterionic detergents are Zwittergent.RTM. 3-08, 3-10, 3-12 and 3-14 (the 3-14 compound of Calbiochem.RTM. is synonym for n-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate). The person of skill in the art is able to distinguish which detergents are suitable, as those would provide results with well-separated antigens that may easily be quantified. Unsuitable detergents that would disrupt or even destroy the antigens may just as easily be distinguished, since these would not give proper separated peaks in the chromatograms.

The problem to be solved was to provide an accurate, rapid and robust method, that would be applicable for high-throughput, and that would not have the disadvantages as found in the art. The inventors of the present invention have now found that RP-HPLC can nevertheless be used for this purpose and that such columns can be used if the temperature of the column was raised up to (but not including or above) the temperature with which the column material can no longer be used. The inventors have found that the hemagglutinin antigen, and especially the main determinant HA1 is separated extremely well from the other proteins present in the preparation. This now enables the skilled person to measure the peak of the separated protein in an RP-HPLC chromatogram and to determine the amount present in the preparation, either by comparing it to other (known) values or to internal standards. Preferably, the elution is performed at a temperature above room temperature, typically above approximately 25.degree. C. The inventors have found that values above room temperature are well suited for this purpose. It is also found that temperatures between about 50.degree. C. and about 70.degree. C. are even more suitable for said purpose. Thus, in a preferred embodiment, the invention relates to a method according to the invention, wherein said elution is performed at a temperature between about 25.degree. C. and about 70.degree. C., more preferably between about 40.degree. C. and about 70.degree. C., and even more preferably between about 50.degree. C. and about 70.degree. C., most preferably between about 60.degree. C. and about 70.degree. C. The best performance was detected at about 60.degree. C. and about 70.degree. C., which latter value is close to the temperature with which the column material can no longer be applied for proper separation purposes. It is thus also part of the invention to perform the methods of the invention up to the highest temperature possible before the column material does no longer allow proper separation. Typically, most elutions were performed at a temperature of about 65.degree. C. Suitable column materials that are typically used are polymer-based materials. Silica-based materials are less suitable, since they generally do not allow elution at high temperatures. The person skilled in the art of RP-HPLC can easily determine to what temperature certain column materials can be raised before they become useless for said purpose. So, in a highly preferred embodiment, the invention relates to a method according to the invention, wherein said elution is performed at a temperature of approximately 60.degree. C., approximately 65.degree. C. or approximately 70.degree. C.

It is to be understood that typical methods of RP-HPLC technology have been applied, and that a person skilled in the art of (RP-)HPLC is well aware of minor adjustments that would not alter the results to be obtained, such as different measurements at other suitable wavelengths or by the use of other column material that would not severely alter the results obtained by the present invention.

As mentioned infra, it is a well-known fact in the art that the mature influenza antigen HA0 is processed to the sub-fragments HA1, and HA2, upon cleaving with for example trypsin. Since the methods according to the invention use the separation in RP-HPLC such that the HA1 peak is measured for proper and accurate determination of the titer, it is preferred to have full cleavage of the mature protein. This can be achieved by a further step in which a protease compound is added that cleaves most if not all un-cleaved mature protein into the two desired sub-fragments. Typically, but not necessarily, the compound trypsin is used for this purpose. Thus, the invention also relates to a method according to the invention, comprising the further step of incubating the antigen preparation with a protease such as trypsin. This step is suitable for cleaving most if not all remaining un-cleaved mature forms of the HA antigen. Since the trypsin component is preferably removed from the solution before analysis, it is preferred to have the protease such as trypsin present on beads, preferably agarose beads. These beads can easily be removed by centrifugation, after the trypsin has cleaved most, if not all, HA0 into its separate subunits. Clearly, in another setting, one could choose to add trypsin inhibitors after the trypsin has cleaved all HA0, in which case the use of beads is not necessary.

Importantly, it was also noticed by the inventors that upon re-addition of DTT after alkylation, the HA1 recovery seemed to be 6 to 10% higher than after reduction alone. Thus, in one preferred embodiment, a further step is included, wherein the reducing agent is added after alkylation of the reduced antigen in the sample preparation procedure.

The methods of the present invention now enable one of skill in the art to separate HA1 from other proteins in a very robust, rapid and accurate way. The RP-HPLC chromatograms that are produced in machines applied for the methods of the present invention can also be used to determine the peak values of the separated proteins. Since these can be compared to known values of known antigens or to internal values used by the person carrying out the method, one is now able to accurately determine the amount of antigen present in the starting material. Thus, the present invention also relates to a method for quantifying the HA titer of an HA antigen preparation, said method comprising the method of separating the HA according to the invention, with the further step of measuring the peak area of the eluted antigen in a chromatogram resulting from the elution step. Preferably, said method of quantifying is applied for influenza antigens; a preferred embodiment relates to a quantification method according to the invention, wherein said HA antigen is of an influenza A virus.

Claim 1 of 19 Claims

1. A method for separating hemagglutinin (HA) HA1 and HA2 antigens from an influenza A virus or an influenza B virus, said method comprising: applying a reduced and derivatized antigen preparation comprising solubilized HA1 and HA2 antigens and a detergent in a pH controlled solution to a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) column, wherein a mature form of HA in an antigen preparation has been cleaved by a protease into HA1 and HA2 prior to applying the antigen preparation to the RP-HPLC column; and eluting the HA1 and HA2 antigens from the column with an ion pairing agent in an organic mobile phase, wherein the HA antigens are derivatized with an alkylating agent, wherein the elution results in separation of HA1from HA2, and wherein the elution is performed at a temperature of between about 50.degree. C. and about 70.degree. C.
 

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