Patent Application
20060051742
The invention relates generally to the field of vaccine manufacturing. More in particular, the invention relates to the production of influenza vaccines and the determination of antigen concentration in influenza virus preparations.
Influenza viruses are generally divided into three types: A, B, and C, based on the antigenic differences between their nucleoprotein and matrix protein antigens. Influenza A viruses are further divided into subtypes depending on the antigenic nature of the two major viral surface proteins, the hemagglutinin (HA) and neuramimidase (NA) proteins. Currently, 15 subtypes of HA are known (Lamb and Krug, 2001). Both HA and NA carry antigenic epitopes. Antibodies that are raised against HA and NA are associated with resistance to infection and/or illness in humans and animals. The efficacy of a vaccination against influenza is largely determined by the amount of immunogenic HA in a vaccine (Wright and Webster, 2001).
For several decades, the HA content of influenza whole-virus and split vaccines derived therefrom, has been assayed using Single Radial Immunodiffusion (SRID). In SRID, influenza virions are disrupted by detergent, and submitted to immunodiffusion for three days at room temperature in antibody-loaded agarose gels. Upon gel staining, the precipitation zone diameters of antigen-antibody complexes are measured, and the antigen content of virus preparations of a certain subtype is calculated by using a calibration curve obtained with a whole virus reference batch of this subtype (NIBSC, Hertfordshire, UK) with a known HA content (Wood et al. 1977).
However, this SRID assay has a number of disadvantages. Apart from being time consuming, laborious and not leaving room for very high throughput (Wood et al. 1977), the quantification of HA by SRID was shown to be inaccurate when analyzing split vaccines or subunit vaccines (Johannsen et al. 1985). In addition, the virus sample environmental background (its pH and ionic strength) and the choice of detergent for disintegrating the influenza virus and its HA were shown to affect the determination of the HA titer (Willkommen et al. 1983; Bizhanov et al. 1988). Despite all shortcomings of the SRID assay, and calls from experts in the field that in addition to the SRID assay a physico-chemical quantification method be used for the quantification of HA (Pereira, 1973; Johannsen et al. 1985), immunodiffusion techniques are still the only methods approved by regulatory authorities for the evaluation of influenza vaccines.
A Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method to separate influenza virus components has been described (Phelan and Cohen, 1983). Viral proteins were solubilized and denatured in guanidine-HCl, and reduced by incubation with dithiotreitol (DTT) for several hours at room temperature. It is well known that, under denaturing conditions upon reduction, mature and activated HA0 falls apart into the relatively hydrophilic subunit HA1 and the hydrophobic subunit HA2, the latter still containing the transmembrane domain of the original HA0. Subsequently, analysis was performed by RP-HPLC at room temperature on an (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, and the recovery was low and not quantitative, presumably due to aggregation of the virus components and/or nonspecific adsorption to the HPLC system/column. 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.
Kemp et al. (1980) also discloses a method for separating influenza HA using RP-HPLC: radiolabeled tryptic glycopeptides (small parts) of HA are pre-isolated from SDS/PAGE gels and subsequently analyzed by HPLC. The method disclosed by Kemp et al. has the disadvantage of not being suitable for a high-throughput system, because the isolation from gel renders the method rather laborious. Moreover, the chromatographs clearly indicate the poor resolution of the peaks, overlapping with numerous other viral peaks, which makes that the method cannot be used for quantitative purposes. The isolation of numerous bands related to different peptides of different size from gel makes that the method is not suitable for very accurate quantification and repeatability. Moreover, the method of Kemp et al. is not suitable for real-life (non-radiolabeled) samples as the radiolabel is detected, and not suitable for crude sample analyses.
In yet another study (Van der Zee et al. 1983) a method has been disclosed for the purification of Sendai virus envelope proteins using RP-HPLC. Although Van der Zee et al. state that some proteins could be recovered in pure form; this was only assessed by SDS/PAGE, which method is not a very accurate means to show purity of a sample. The chromatograms show that resolution is poor: this indicates that any accurate quantification, based on the HPLC chromatograms is not possible using the purification method disclosed. Moreover, it seems that the detergent interferes with the peak of interest. Also, carry-over of proteins from one analysis to the other is significant. In general, it is clear that the art does not disclose methods and means for an accurate determination of HA concentration in either crude or purified HA samples.
Clearly, a strong need exists for a robust, accurate and fast method for reliable separation and quantification of HA in upstream- and downstream-process preparations, as well as for final vaccine formulations.
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.
The invention also relates to methods for quantifying the HA titer of an HA antigen preparation, the 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.
The invention in particular relates to the separation and quantification of HA of influenza B viruses.
Here, a novel separation and quantification assay for the determination of hemagglutinin (HA) concentration by 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 derivativization 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.
One preferred embodiment of the invention relates to a method according to the invention, wherein the elution is performed at a temperature of between about 25° C. and about 70° C., preferably between about 40° C. and about 70° C., and more preferably between about 50° C. and about 70° C., and most preferably between about 60° C. and 70° C. In another preferred embodiment, the method comprises a step wherein the antigens are cleaved by a protease, such as trypsin.
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° 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 Neuramimidase (“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 HA antigens, the 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 RP-HPLC column; and eluting the HA antigens from the column with an ion pairing agent in an organic mobile phase.
In one embodiment, 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® (Calbiochem-Novabiochem Corporation), 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 dithiotreitol (DTT) to a concentration of, for example, 25 mM, although other concentrations may also be used, and reduction takes place for about ten minutes at about 90° 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 iodacetamide (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; alkyl sulfonates, 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° 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 may be as high as 70° C., but preferably around 65° 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° 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° 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 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 the HA is of an influenza virus or a measles virus. Preferably, the influenza virus is an influenza A virus or an influenza B virus. Also preferred are methods according to the invention wherein the 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 dithiotreitol (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® 3-08, 3-10, 3-12 and 3-14 (the 3-14 compound of CALBIOCHEM® is synonym for n-Tetradecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate). 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 hereof 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° 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° C. and about 70° C. are even more suitable for the purpose. Thus, in a preferred embodiment, the invention relates to a method according to the invention, wherein the elution is performed at a temperature between about 25° C. and about 70° C., more preferably between about 40° C. and about 70° C., and even more preferably between about 50° C. and about 70° C., most preferably between about 60° C. and about 70° C. The best performance was detected at about 60° C. and about 70° 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° 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 the purpose. So, in a highly preferred embodiment, the invention relates to a method according to the invention, wherein the elution is performed at a temperature of approximately 60° C., approximately 65° C. or approximately 70° 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 herein, it is a well-known fact in the art that the mature influenza antigen HA0 is processed into 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. One could also choose to add trypsin inhibitors after the trypsin has cleaved all HA0, in which case the use of beads is unnecessary.
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, the 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, the method of quantifying is applied for influenza antigens; a preferred embodiment relates to a quantification method according to the invention, wherein the HA antigen is of an influenza A virus.
The invention is further explained with the aid of the following illustrative Examples.
The following Influenza A antigens have been used herein (NIBSC-reference numbers underlined):
All influenza antigens were obtained from the National Institute for Biological Standards and Control (NIBSC, Hertfordshire, United Kingdom). The antigen A/Panama/2007/99 (D953-043F) was also produced using PER.C6® cell-based technology.
The egg-derived influenza antigen preparation A/Panama/2007/99 (Resvir-17; H3N2) from NIBSC and the same antigen produced on PER.C6®-based technology, were analyzed on Reversed Phase-HPLC (RP-HPLC).
The production of antigen produced on PER.C6® cells was performed as follows: PER.C6® cells (as represented by the human embryonic retina (HER) cells under ECACC no. 96022940 deposited with the European Collection of Cell Cultures (ECACC) at the Centre for Applied Microbiology and Research (CAMR), Salisbury, Wiltshire, UK, see, e.g., U.S. Pat. No. 5,994,128 to Fallaux et al.) were cultured in a bioreactor (37° C., DO=50%, pH 7.3) until a viable cell density of 1×106 cells/ml was accomplished. The cells were infected with influenza viruses of the strain Resvir-17 (H3N2) (35° C., with a multiplicity of infection of 1×10−4) in the presence of 3 μg/ml trypsin/EDTA. The infection was continued for five days. The bioreactor content was then treated with 10 U/ml benzonase (Merck) for 30 minutes at 37° C. This was followed by clarification with a 3.0 μm filter (Clarigard, Millipore) and a ten-fold concentration step, using tangential flow filtration (Hollow-fiber module, Amersham). Subsequently, the product was applied on sucrose gradient from 10 to 42% in PBS and centrifuged for two hours at 22,000 rpm in an ultracentrifuge (Beckman). The virus band was visible by the eye and was collected using a syringe. This material was used for development of the HPLC method.
Both batches of Resvir-17 antigen were disintegrated by addition of SDS (Gibco BRL) to a final concentration of 1% (w/v), and reduced with 60 mM DTT in 0.15 M Tris, pH 8.0, for 30 minutes at 65° C. After cooling down, reduced proteins were alkylated by incubation with iodacetamide (IAA, final concentration of approximately 106 mM) at 37° C. for 45 minutes in the dark. This alkylation step prevents the released proteins with free reactive sulfhydryl groups (e.g., HA1, HA2, and NA) from associating with each other.
Analysis was performed on an Agilent 1100 HPLC system with 900 μl loop injector, using a polystyrene dimethylbenzene POROS R1/10 (2.1×100 mm) Reversed Phase column (Applied Biosystems), and the gradient profile described in Table 1. Proteins were detected with a multiple wavelength detector at 215 nm.
Between 50-300 μl of sample was injected (approximately 10 μg HA as determined by SRID), and RP-HPLC was performed with a flow of 0.8 ml/minute and at a column temperature of 70° C.
The RP-HPLC assay according to the present invention for quantification of the HA titer in influenza virus preparations is based on measuring the peak area of its subunit HA1. The protein is solubilized upon addition by detergent, submitted to reduction/alkylation with DTT/IAA (respectively), and subsequently analyzed utilizing the RP-HPLC procedure according to the schedule depicted in Table 1. As a consequence, a crucial parameter of the assay is the selectivity, i.e., the resolution between the HA1 peak and other virus-derived material in a Reversed Phase chromatogram. The person skilled in the art is aware of the fact that the organic mobile phase may be performed with different agents. Typically, acetonitrile is used as solvent B (see Table 1). Other solvents B that may be used are methanol, isopropanol and ethanol. As part of solution A and B (see Table 1) an anionic or cationic ion-pairing agent is typically used. Examples of anionic ion-pairing agents that may be used in the methods of the present invention are trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA) and the like. Examples of cationic ion-pairing agents that may be used in the methods of the present invention are tetramethylammonium chloride, tetrabutylammonium chloride, and triethylamine.
The selectivity of the assay was explored first by analyzing formaldehyde-inactivated influenza A subtype Resvir-17 (H3N2) produced in chicken eggs at the NIBSC (
The results of the silver stained SDS-PAGE gel are shown in
PER.C6®-produced Resvir-17 antigen material was also analyzed by RP-HPLC (
Consequently, these data indicate that the assay selectivity, i.e. the separation of HA1 with the other viral components in both egg- and PER.C6®-derived H3N2 Resvir-17 antigens, was excellent.
Further, it was investigated whether the RP-HPLC assay was also applicable for hemagglutinins from other influenza A subtypes. Hence, the selectivity of the assay with two other influenza A subtypes, A/Duck/Sing (H5N3) and A/New Caledonia (H1N1) was determined.
First, an RP-HPLC was performed on egg-derived and formaldehyde-treated H5N3 from A/Duck/Sing. For this an amount corresponding to 3.0 μg HA was injected. Further procedures were as described in Example 1, except that instead of SDS, ZWITTERGENT® 1% (w/v) was used as the detergent. In
A graph of the RP-HPLC of egg-derived, reduced and derivatized influenza A subtype H1N1 (A/New Caledonia) is shown in
Taken together, these data demonstrate that the assay selectivity for quantification of HA1 is excellent, and in addition, that the RP-HPLC assay is not specific for a particular influenza A subtype, but that it can be applied broadly for different types of influenza viruses.
One of the key criteria of an analytical procedure is linearity, the ability (within a certain range) to obtain test results, which are directly proportional to the concentration (amount) of analyte in the sample. Assay linearity was studied with egg-derived, formaldehyde-inactivated, reduced and alkylated influenza antigen from A/Panama/2007/99 (A/Resvir-17; H3N2). Increasing concentrations of HA were injected on the RP-HPLC system with a constant injection volume of 200 μl, and subsequently plotted versus the measured area of the HA1 peak, resulting in a calibration curve as shown in
As an important conclusion, as discussed infra, the HA titer of formaldehyde-inactivated influenza samples like the one used above cannot be determined to the highest possible accuracy. Moreover, in the linearity study just discussed sample preparation had not been optimal. Taking these two points into account, linearity was, therefore, also studied with a PER.C6®-derived but BPL-inactivated A/Resvir-17 sample. The data showed that for the BPL-inactivated influenza A/Resvir-17 sample good assay linearity could be achieved (
Assay precision was studied by analyzing six injections of a reduced and alkylated sample of A/New Caledonia (H1N1) at a relatively low concentration (0.65 μg HA per injection). In addition, precision was also explored by injecting four independently reduced and alkylated samples with a relatively high HA titer (about 3 μg HA per injection). Results are shown in Tables 2 and 3, and demonstrate that the precision was good for both sets of samples with CVs below 10%.
The effect of column temperature on the assay was also studied. In this respect, egg-derived Resvir-17 (H3N2) antigen was reduced and alkylated as described above, except that these reactions were conducted under non-buffered conditions. Subsequently, samples (approximately 4.3 μg HA per injection) were analyzed at the following column temperatures: 25° C., 40° C., 50° C., 60° C., and 70° C., using the acetonitrile gradient in an organic mobile phase as described in Table 4.
In
In a subsequent experiment, the effect of column temperature on RP-HPLC of influenza A/Resvir-17 was again investigated, and at each test temperature samples were analyzed in triplicate. In addition, influenza A/New Caledonia (H1N1) was taken along (single injection at each temperature). As illustrated in
It was further investigated whether the RP-HPLC assay was also applicable for hemagglutinin from influenza A subtype H7N7. Hence, the selectivity of the assay with formaldehyde-inactivated subtype A/Equine/Prague/56 (H7N7) was determined. Sample preparation and further procedures were generally as described in Example 1 (1% SDS as detergent, reduction with 65 mM DTT, 65° C., 30 minutes, alkylation with IAA 116 mM, 37° C., 45 minutes in the dark). In
Taken together, as mentioned under Example 2, these data demonstrate that the assay selectivity for quantification of HA1 is excellent, and in addition, that the RP-HPLC assay is not specific for a particular influenza A subtype, but that it can be applied broadly for different types of influenza viruses.
As noticed above, HA in both formaldehyde-inactivated egg-based and beta-propiolactone-inactivated PER.C6®-derived influenza A/Resvir-17 (H3N2) did not turn out to be fully cleavable upon reduction (59 mM DTT, 30 minutes at 65° C.). This is a highly undesired situation, as the most accurate HA quantification by RP-HPLC requires a full cleavage of HA into HA1 and HA2. It was explored whether this could be accomplished by applying more severe reduction conditions. However, the ratio between non-cleaved HA0 and HA1 was not affected by reduction for longer times and/or at higher temperatures (data not shown). This indicated that most likely part of the HA in both preparations had not been cleaved enzymatically, and could therefore never be split upon reduction.
Then, it was investigated whether the cleavage of the residual un-cleaved HA was possible by additional trypsin treatment of both vaccins dissociated in 1% SDS or 1% ZWITTERGENT®. Under these conditions, HA is in its trimeric format, which is susceptible to the trypsin-induced specific cleavage into its subunits, but resistant to further proteolytic breakdown. For this, agarose beads conjugated with trypsin were utilized, which enables one to remove the beads conveniently by centrifugation after digestion, thereby also avoiding possible proteolytic degradation of trypsin-sensitive HA1 during the further sample preparation (e.g., the reduction step). Buffer solution was 134 mM Tris-HCL, pH 8.0 and reduction was performed with DTT at 100° C. for ten minutes. Alkylation was performed by IAA treatment as in the previous examples. During trypsin treatment (15, 35, 60 and 120 minutes), samples were rotated in an oven at 37° C. to prevent the trypsin beads from precipitating in the sample tubes.
Pre-treatment of BPL-inactivated PER.C6®-based Resvir-17 in 1% ZWITTERGENT® resulted in the disappearance of the residual amount of un-cleaved HA0, while further breakdown of HA1 and/or HA2 was not detected (left panel,
In contrast, the situation for formaldehyde-inactivated egg-based A/Resvir-17 was quite different: in the presence of either SDS or ZWITTERGENT® cleavage of HA0 by additional trypsin pre-treatment did not turn out to be possible (data not shown). This was most likely due to the formaldehyde-inactivation treatment (different from the beta-propiolactone treatment discussed above). Formaldehyde is known to cause irreversible cross-linking of proteins, which are in a complex, like the disulfide-linked HA1 and HA2. Consequently, it is preferred to use trypsin to ensure a full cleavage of HA0, but if trypsin is used it should be used in a detergent such as ZWITTERGENT®, rather than SDS, while trypsin should preferably be used on samples that were previously inactivated through an inactivating agent such as BPL, rather than cross-linking inactivating agents such as formaldehyde.
Initially, influenza samples were reduced and alkylated under non-buffered conditions. When injected immediately after the non-buffered reduction and alkylation reactions (1% ZWITTERGENT®; reduction 59 mM DTT, 65° C. 30 minutes; alkylation 106 mM IAA, 37° C. 45 minutes in the dark), RP-HPLC of PER.C6®-based A/Resvir-17 HA resulted in a sharp HA1 peak, eluting at approximately 8.9 minutes in the chromatogram (
On the other hand, the observed HA1 peak deformation in time might also be caused by the presence of residual amounts of IAA, as it is generally known that IAA may give rise to relatively strong adverse effects on the integrity of proteins. Then, the effect of omission of the alkylation step after reduction was studied. This however, did not have a significant effect on HA1 recovery, but, interestingly, regarding the HA1 peak shape, samples proved to be far more stable in time (
To distinguish whether the apparent HA1 instability was due to the absence of reducing circumstances or to possible disadvantageous side effects of the residual amount of IAA chemically modifying the protein, the HA1 stability was also monitored after reduction (without subsequent alkylation) at various DTT concentrations before and after storage for 18 hours at 4° C. As can be seen in
Unexpectedly, a different (putative) HA1-peak instability was observed: after 18 hours at 4° C. and at low DTT-concentrations (1-4 mM) a small, but significant peak was discernible in the tailing part of the original HA1 peak (
The stability of the HA1 peak area was monitored in triplicate for both a reduced/alkylated/DTT treated sample and an only reduced PER.C6®-based influenza A/Resvir-17 sample before and after storage for 20 hours at 4° C. Notably, reduction was carried out at a DTT concentration of 25 mM, and after the alkylation reaction (as for half of the samples) DTT was re-added to a final concentration of 25 mM, to prevent any HA1 peak deformation. It turned out that, unlike the experiment of
Above, it has been indicated that trypsin treatment ensures a full cleavage of HA into its subunits HA1 and HA2. It should be noted that if the cleavage, due to for instance cellular proteases may be complete and that an extra trypsin treatment may be omitted. Nevertheless, to ensure that all HA0 is cleaved, it is preferred to add the additional trypsin step. An experiment was designed to explore the effect of increasing concentrations of trypsin (preferably present on beads) on the ultimate recovery of HA1 from three samples of PER.C6®-based influenza A/New Caledonia, which differed in the amount of virus and, hence, in HA content. It turned out that for each of the three samples addition of 120 mU trypsin beads, and subsequent incubation for 30 minutes at 37° C. resulted in optimal HA1 recoveries (
As disclosed herein, it was demonstrated that the assay selectivity, linearity and precision of the method according to the invention were good. To explore whether the RP-HPLC assay according to the invention would provide for a proper alternative for the cumbersome and slow SRID assay, results were compared between the two assays. In Table 8, a first comparison was made between both assays for a number of A/Resvir-17 samples. It must be noted that trypsin pretreatment and alkylation were not included as standard steps. Six different samples were compared, whereas the concentration of the samples A, C and D were determined in triplicate by HPLC (e.g., A1, A2, A3). The Table shows that the HA titers obtained by RP-HPLC closely resembled those acquired by SRID.
Interestingly, the data also demonstrated that formaldehyde treatment of A/Resvir-17 resulted in a greatly reduced HA1 peak as compared to the same, but BPL-inactivated batch (Table 8; compare samples E and F), supporting earlier conclusions that HA quantification in formaldehyde-inactivated influenza batches is far from accurate.
Subsequently, these comparative studies between RP-HPLC and SRID were repeated in more detail for a series of A/New Caledonia samples. As shown in Table 9, the HPLC data agreed well with the SRID-based titres. Consequently, it was thus established that the RP-HPLC assay is accurate, and represents a good alternative for the SRID assay to quantify the HA concentration in influenza virus containing batches. This is certainly the case when taking into account that the HPLC assay precision is better than the precision attained by SRID (Table 9, see RSD values for sample D).
From a process development point of view, another important application of the assay would be measuring the HA concentration in crude culture supernatants of cells infected with influenza virus. To explore the feasibility of this potential application we studied the assay selectivity with regard to the sample matrix, i.e., conditioned growth medium of PER.C6®. When analyzing a sample of a crude culture supernatant of PER.C6® grown in AEM medium and infected with influenza A/Resvir-17 without any sample treatment other than reduction alone, a rather complex chromatogram was recorded (
It was investigated whether the RP-HPLC assay was also applicable for hemagglutinin from B-strains. Hence, the selectivity of the assay with PER.C6-produced influenza B/Jiangsu, and BPL-inactivated egg-based B/Guangdong was determined. Sample preparation and further procedures were as described for A-strains. The acetonitrile gradient was slightly changed from the method for A-strains (Table 10). In
In
| Number | Date | Country | Kind |
|---|---|---|---|
| 04/50318 | Mar 2004 | EP | regional |
This application is a continuation-in-part of PCT International Patent Application No. PCT/EP2005/050957, filed on Mar. 3, 2005, designating the United States of America, which itself claims priority to PCT International Patent Application No. PCT/EP2004/050318, filed on Mar. 17, 2004, designating the United States of America, the contents of the entirety of both of which are incorporated herein by this reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP05/50957 | Mar 2005 | US |
| Child | 11119631 | May 2005 | US |
