Lyophilised antigen composition

a technology of lyophilised antigen and composition, which is applied in the direction of drug compositions, immunological disorders, antibody medical ingredients, etc., can solve the problems of increasing the complexity of preparation of components, distribution and formulation of vaccine compositions, and difficulty in liquid vaccine formulations, so as to increase the solubility of lyophilised antigens, increase and improve the solubility of antigens. the effect of cost benefits

Inactive Publication Date: 2009-02-05
GLAXOSMITHKLINE BIOLOGICALS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017]Whilst not wishing to be bound by theory it is thought that providing the antigen and the TLR9 agonist together provides a component that is more stable than simply the addition of the TLR9 to a liquid formulation of MPL and QS21.
[0018]The present invention provides the advantage that where the antigen and TLR9 agonist are reconstituted with WFI one is able to provide only one vial with lyophilized formulation in it. Furthermore, where the antigen and the TRL9 agonist are to be reconstituted with a liquid formulation such as a liquid adjuvant formulation then it is advantageous to be able to provide only two vials of components (rather than three). This in turn has cost benefits, whilst providing a product suitable for use a vaccine once reconstituted.
[0019]Furthermore, the present inventors have found that the co-lyophilisation of CpG with antigens which would not have an overall positive charge in the reconstitution buffer may increase the solubility of those antigens on reconstitution with either water for injection or liquid adjuvant. Therefore the present invention also provides a method to increase the solubility of a lyophilised antigen on reconstitution where the antigen would not have a net positive charge in the reconstitution buffer comprising the step of co-lyophilising a TLR9 agonist, preferably an immunostimulatory oligonucleotide and more preferably a CpG oligonucleotide with the antigen. The present invention also provides for the use of a TLR9 agonist, preferably an immunostimulatory oligonucleotide and more preferably a CpG oligonucleotide to increase the solubility of a lyophilised non-positively charged antigen on reconstitution. By “non-positively charged” is meant that the overall charge of the protein is not positive. The protein may contain both positive and negative charges, but the overall charge of the protein is either neutral or negative.

Problems solved by technology

However the inclusion of adjuvants into a vaccine or immunogenic composition increases the complexity of preparation of the components as well as the complexity of distribution and formulation of the vaccine composition.
The antigen may, for example not be stable when stored for prolonged periods at this pH.
This may make them difficult to incorporate in liquid vaccine formulations because they are dissimilar to other components in the formulations.
As discussed this may cause precipitation and / or long term stability problems.
However, an increasing number of components in an increasing number of vials leads to increased costs, waste and importantly to an increase in the possibility of mistakes during constitution.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Freeze Drying of a CpG Oligonucleotide and CPC-P501S as Antigen

[0159]The antigen used was CPC-P501S. This antigen is shown in FIG. 1 diagrammatically, in which the section showing TM2 to TM12 represents the P501S antigen; the oval shapes on the left hand side represent the CPC fusion partners and the His tail is shown on the right hand side.

[0160]The antigen was produced with a His tag as shown in S. cerevisiae and then made to a concentration of 700 μg / ml using a buffer of Tris (5 mM pH7.5) and Tween80 (0.3%).

[0161]To prepare the final bulk, sucrose (35%) was added to water for injection to reach a final concentration of 6.3%. Tris (1M pH8.8) was then added, followed by Tween 80 (25%) to reach a final concentration of 0.2%. This mixture was magnetically stirred for 5 minutes at room temperature. CPC-P501S was added and the mixture was magnetically stirred for 4 minutes at room temperature. A CpG oligo of SEQ ID No:4 was then added, and the resulting mixture magnetically stirred for...

example 2

Freeze Drying of a CpG Oligonucleotide and Mage-3 as Antigen

[0168]The antigen used was a portion of the protein D protein linked to MAGE-3, which in turn was linked to a His tail for ease of purification PD-Mage3-His (see FIG. 5: SEQ ID NO: 13).

[0169]The purified bulk antigen was produced with a His tag in E. coli and then made to a concentration of 750 μg / ml using a buffer of NaH2PO4.2H2O / K2HPO4.2H2O (2 mM) and Tween80 at approximately 0.2% v / v (theoreitical) pH7.5.

[0170]To prepare the final bulk, sucrose (30%) was added to water for injection to give a final concentration of 3.15%. NaH2PO4.2H2O / K2HPO4.2H2O (100 mM pH7.5) was then added to give a final PO4 concentration of 5 mM taking into account the phosphate found in the antigen buffer. Tween 80 (3%) was also added to give a final concentration of 0.15%, taking into account the Tween found in the antigen buffer. This mixture was magnetically stirred for between 5 and 15 minutes at room temperature. PD-Mage3-His was added (750 μg...

example 3

Impact of CpG on Antigen Solubility Following Reconstitution

[0179]1. WT1 is a protein originally found to be overexpressed in paediatric kidney cancer, Wilm's Tumor. The candidate antigen used in the present case uses nearly the full length protein as antigen. The WT1-A10 protein is a 292 AA recombinant fusion protein expressed in E. coli consisting of a 12mer truncated tat sequence (leader sequence) and amino acids number 2-281 of the WT1 sequence. After lyophilisation alone, this antigen precipates if reconstituted with adjuvant system A due to its isoelectric point (5.85 to 7.5) which is close to the pH of adjuvant system A (6.1) and the presence of sodium chloride in adjuvant system A.

[0180]Two formulations of WT1-A10 were prepared. The reconstituted dose contained 400 μg / ml of WT1-A10 antigen, 10% sucrose, 100 mM Tris, and 0.2% Tween 80, plus or minus 840 μg / ml CpG.

[0181]Both formulations were reconstituted with 500 μl of adjuvant system A. The resulting liquid was centrifuged ...

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Abstract

The present invention provides lyophilised compositions comprising an antigen and a Toll-like receptor (TLR) 9 agonist. Such compositions may be reconstituted into immunogenic compositions for use in vaccination with a carrier selected from the group of particulate carriers consisting of liposomes, mineral salts, emulsions, polymers and ISCOMs. Methods of making immunogenic compositions from the lyophilised compositions of the invention and use of the same in immunisation are also herein provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to PCT / EP2007 / 055037 filed May 24, 2007, GB0723044.4 filed Nov. 23, 2007 and GB0723900.7 filed Dec. 6, 2007, all of which are incorporated herein in their entirety.FIELD OF THE INVENTION[0002]The present invention relates to improved antigenic compositions and methods of using the same to make immunogenic compositions. In particular the present invention relates to lyophilised compositions comprising an antigen and a Toll-like receptor (TLR) 9 agonist. Such compositions may be reconstituted into immunogenic compositions for use in vaccination with a carrier selected from the group of particulate carriers consisting of liposomes, mineral salts, emulsions, polymers and ISCOMs. Methods of making immunogenic compositions from the lyophilised compositions of the invention and use of the same in immunisation are also part of the present invention.BACKGROUND TO THE INVENTION[0003]Adjuvants are sometimes used to i...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K9/127A61K39/15A61P37/04A61K39/00
CPCA61K31/7088A61K39/0011A61K39/102A61K39/21A61K2039/545C12N2310/17A61K2039/55555A61K2039/55561C07K2319/00C12N15/117A61K2039/55544A61K39/12A61P31/18A61P35/00A61P37/00A61P37/04A61P43/00A61K39/001153A61K39/39
Inventor LEMOINE, DOMINIQUE INGRID
Owner GLAXOSMITHKLINE BIOLOGICALS SA
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