Method for preparing liposome-based constructs

Abstract

The present invention relates to method for preparing liposome-based constructs comprising a peptide, particularly an antigenic peptide, of interest modified through hydrophobic moieties reconstituted in liposomes and to the antigenic constructs obtained with said method. The invention further relates to the use of said constructs for the therapeutic and diagnostic use in the treatment of diseases and disorders, which are caused by or associated with proteopathy such as Alzheimer's Disease.

Claims

1. A method of preparing a liposome-based construct comprising a peptide modified through hydrophobic moieties reconstituted in a liposome, comprising the steps of (i) preparing liposomes in a solution; (ii) preparing a modified peptide by adding to the N- and/or C-terminus of the peptide molecule at least one hydrophobic moiety; (iii) solubilizing the modified peptide in the presence of a surfactant such that the modified peptide is in a micellar form; (iv) loading the prepared liposomes of step (i) by adding the solubilized modified peptide to the prepared liposomes and diluting the solubilized modified peptide below a critical micellar concentration of the surfactant such that the micellar form of the modified peptide is disrupted thereby driving integration of the hydrophobic moieties of the modified peptide into an external layer of the preformed liposomes such that at least 72%, 80%, 90%, or 100% of said peptide is present on the outer surface of the liposome.

2. The method of claim 1, further comprising loading the preformed liposomes with an adjuvant, wherein the adjuvant loading is carried out (a) prior to, (b) together with, or (c) after the loading of the liposomes with the diluted solubilized antigenic peptide.

3. The method of claim 1, wherein the peptide is modified by: (a) addition of a fatty acid, a triglyceride, a diglyceride, a steroid, a sphingolipid, a glycolipid, a phospholipid, or a combination thereof; (b) at least 2 palmitoylated amino acid residues covalently attached to the N- and C-terminus of the peptide, respectively; or (c) at least 4 palmitoylated amino acid residues, two of which are covalently attached to the N- and C-terminus of the peptide, respectively.

Description

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES

(1) FIG. 1 shows a flow chart of L16 process.

(2) FIG. 2 shows analysis of anti-TAU5-20 [pY18] IgG antibodies in the plasma of C5BL/6 mice after receiving ACI-33 vaccines, either manufactured with thin film method (Process A) or with process L15. A pre-bleeding was done at 7 days followed by day 7, 21 and 35 after the first immunization. Results are expressed as meanstandard deviation obtained in groups of 10 mice.

(3) FIG. 3 shows analysis of anti-TAU396-408 [pS396/pS404] IgG antibodies in the plasma of C5BL/6 mice after receiving ACI-35 vaccines, either manufactured with thin film method (Process A) or with process L15. A pre-bleeding was done at 7 days followed by day 7, 21 and 35 after the first immunization. Results are expressed as meanstandard deviation obtained in groups of 10 mice.

(4) FIG. 4 shows analysis of anti-A IgG antibodies in the plasma of C5BL/6 mice after receiving PaI 1-15 vaccines, either manufactured with process D (ACI-24 process D#2) or with process L15. A pre-bleeding was done at 7 days followed by day 7, 21 and 35 after the first immunization. Results are expressed as meanstandard deviation obtained in groups of 10 mice.

(5) FIG. 5 shows analysis of anti-A IgG antibodies in the plasma of C5BL/6 mice after receiving PaI 1-15 vaccines, either manufactured with the process D (ACI-24 process D#1) with the process L15 (ACI-24 process L15 A; L15B and L15C) or with the ACI-24 process L16. A pre-bleeding was done at 7 days followed by day 7, 21 and 35 after the first immunization. Results are expressed as meanstandard deviation obtained in groups of 10 mice.

(6) FIG. 6a shows analysis of the anti-T8 IgG antibody titers in the plasma of C5BL/6 mice after receiving ACI-41(peptides T8 and T9) vaccines manufactured with L15 process. A pre-bleeding was done at 7 days followed by day 7, 21 and 35 after the first immunization. Results are expressed as meanstandard deviation obtained in groups of 10 mice.

(7) FIG. 6b shows analysis of the anti-T9 IgG antibody titers in the plasma of C5BL/6 mice after receiving ACI-41(peptides T8 and T9) vaccines manufactured with L15 process. A pre-bleeding was done at 7 days followed by day 7, 21 and 35 after the first immunization. Results are expressed as meanstandard deviation obtained in groups of 10 mice.

(8) FIG. 7 shows bicinchoninic acid protein quantification assay (BCA) standard curve of Ac1-15 peptide after solubilizing in either PBS or 2.25% SDS.

(9) FIG. 8 shows Absorbance spectra of ACI-24 with or without BCA reagents, in the presence of either PBS or SDS.

(10) FIG. 9 shows comparison of ACI-24 absorbance spectra upon treatment with BCA reagents and correction for intrinsic liposome absorbance.

(11) FIG. 10 shows absorbance spectra of liposomes lacking peptide (empty liposomes, ACI-24E).

(12) FIG. 11 shows absorbance spectra of liposomes containing only encapsulated Ac1-15 peptide (vaccine ACI-16).

(13) FIG. 12 shows comparison of absorbance spectra for batches of ACI-24 prepared with process D (ACI240908-A) or process L15 (ACI-24-100316-A).

(14) FIG. 13 shows IgG titers at day 7; 7; 21 and 35 after immunization with ACI-35 vaccine generated with either process L15 or L20.

(15) FIG. 14 shows total anti-A titers obtained after 7, 21 or 35 days after injecting either vaccine ACI-17 (process L15 where MPLA is replaced by lipidated CpG adjuvant) or vaccine ACI-18 (process L15 where MPLA is replaced by Pam2CSK4 adjuvant) in mice.

(16) Table 1 to 3 describe batches of L15 process producing PaI 1-15 vaccine. The batches were generated in order to evaluate the physicochemical and in-vivo reproducibility of ACI-24 vaccine generated with process L15: ACI-24-100316-A; ACI-24-100316-B and ACI-24-100316-C. The three vaccines were generated independently.

(17) Table 4 describes a batch of L16 process with normal MPLA concentration producing PaI 1-15 vaccine. The batch was manufactured with process L16 (antigen and adjuvant added after liposome formation)ACI-24-091127-A.

(18) Table 5 describes a batch of L16 process with high MPLA concentration producing PaI 1-15 vaccine. The batch was manufactured with process L16 (antigen and adjuvant added after liposome formation)ACI-24-091127-B.

(19) Table 6 describes a batch of L15 process producing T1 vaccine ACI-33-091127.

(20) Table 7 describes a batch of thin film process producing T1 vaccineACI-33-091808-A.

(21) Table 8 describes a batch of L15 process producing T3 vaccineACI-35-091127.

(22) Table 9 describes a batch of thin film process producing T3 vaccineACI-35-0910820-A.

(23) Table 10 describes a batch of L15 process producing T8/T9 vaccinesACI-41-100531.

(24) Table 11 and 12 describe two independent batches generated by the cross flow ethanol injection method (process D) for producing PaI 1-15 vaccineACI-24 process D#1 and D#2, respectively.

(25) Table 13 shows absorbance values for triplicate analyses of ACI-24-100316-A using the BCA assay.

(26) Table 14 shows summary of analyses results of different ACI-24 processes and batches.

(27) The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practicing the present invention and are not intended to limit the scope of the invention.

(28) The following Examples illustrate the invention.

EXAMPLES

Example 1: Preparation of a Liposome-Based Antigenic Construct (Process L15; L16 and L20)

(29) 1.1. Process L16

(30) 1.1.1 Liposome Preparation:

(31) The phospholipids dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidyl-glycerol (DMPG) and cholesterol (Avanti Polar Lipids. Alabaster, Ala.) were mixed in ethanol (100 ml) at a molar ratio of 9.0; 1.0 and 7.0, respectively. A perfectly clear solution was formed following a continuous agitation at 60 C. for 15 min (see FIG. 1). This lipid mixture was then diluted (17) by injecting the solution (100 ml) in 1600 ml phosphate buffer (PBS) pH 7.4, allowing the formation of multi-layer vesicles (Multilayer Vesiclesstage 1). The resulting preparation was then concentrated by ultrafiltration (Vivaflow 200-100.000 MWCO Polyethersulphone with a flow of 200 ml/min) and the reaction volume was reduced from 1700 ml to 250 ml (Ultrafiltration step). The concentrated solution was further submitted to dialysis in a Vivaflow 200 device where a 10 volume exchange is performed with PBS pH 7.4 (Diafiltration step). The dialysed solution (Multilayer vesiclesstage 2) was then homogenized (7 cycles at 15.000-20.000 Psi) in an Emulsiflex C5 device from Avestin (Homogenization step), followed by 3 cycles of Extrusion (using the same Emulsiflex instrument) through a 0.2 um Polycarbonate membrane with a diameter of 47 mm (Extrusion step). Following those last two sizing steps, unilamelar liposomes with no antigen and adjuvant were formed (Empty liposome preparation).

(32) 1.1.2 Preparation of Adjuvant Solution:

(33) A 765 ug/ml solution of MPLA was prepared in 20 ml PBS pH 7.4 with 1.6% (wt/v) Octyl-Beta-D-Glucopyranoside (B-OG). The resulting solution contained detergent (B-OG) at a concentration above its critical micellar concentration (CMC) of 0.73% (wt/v). This solution was then heated at 60 C. for 30 min and manually injected into the 250 ml preparation of empty liposomes. During this dilution step, the detergent concentration is diluted down 13.5, resulting in a final concentration of 0.12% (wt/v), a concentration of B-OG below its CMC (1.sup.st dilution step). The Liposome solution containing the adjuvant (MPLA) is then diluted (7) by injecting 1450 ml of PBS pH 7.4 (2.sup.nd dilution step).

(34) 1.1.3 Preparation of Peptide Solution:

(35) 1.33 mg/ml of the peptide A-PaI 1-15 was prepared in PBS pH 11.8 (total volume 45 ml) with 2.0% (wt/v) B-OG. The resulting solution comprised a detergent concentration above its critical micellar concentration (CMC) of 0.73% (wt/v). This solution was stirred heated at 60 C. and stirred for 15 min, until a clear solution was formed. The peptide solution (45 ml) was then added to the solution of liposome with adjuvant (1700 ml) and stirred for 1 h at 60 C. resulting in a solution having a final B-OG concentration of (0.05%), which is far below the detergent's CMC (0.73%) (Dilution step). The resulting solution was then concentrated by ultrafiltration (same condition mentioned above) and the final vaccine volume set to 100 ml. The concentrated solution was dialysed by diafiltration, where a 10 volume exchange was performed with PBS pH 7.4.

(36) In a final step, the vaccine solution was sterile filtered through a 0.2 um Polyethersulfone membrane filter (Sartorius 16541-K). Each filter was used to sterile filter 5 ml of vaccine solution into 15 ml Falcon tubes. This last process step is executed in a sterile environment (laminar flow hood).

(37) 1.2. Process L15

(38) Process L15 only differs from process L16 in that the adjuvant (e.g. MPLA) is added together with the lipids in the ethanol solution prior to liposome formation and sizing steps. Therefore, the process of adding adjuvant following liposome formation is suppressed.

(39) 1.3. Process L20

(40) 1.3.1 Liposome Preparation:

(41) The phospholipids dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidyl-glycerol (DMPG) and cholesterol (Avanti Polar Lipids. Alabaster, Ala.) were mixed in ethanol (8.55 ml) at a molar ratio of 9.0; 1.0 and 7.0, respectively. A perfectly clear solution was formed following a continuous agitation at 60 C. for 15 min. MPLA (7.5 mg) was solubilized in 0.45 ml tert-butanol at 60 C. and added to the ethanol solution. This lipid mixture was then diluted (10) by injecting the solution (9.0 ml) in 90 ml phosphate buffer (PBS) pH 7.4, allowing the formation of multi-layer vesicles. The resulting preparation was then extruded through a pack of 3 polycarbonate membranes with a pore size of 0.08 um using an Emulsiflex C5 device from Avestin or a Lipex Extruder from Northern Lipids and diluted with PBS pH 7.4 to a final volume of 425 ml.

(42) 1.3.2 Preparation of Peptide Solution:

(43) 1.33 mg/ml of the TAU peptide T3 was prepared in PBS pH 11.8 (total volume 22.5 ml) with 5.0% (wt/v) B-OG. The resulting solution comprised a detergent concentration above its critical micellar concentration (CMC) of 0.73% (wt/v). This solution was stirred heated at 60 C. and stirred for 15 min, until a clear solution was formed. The peptide solution (22.5 ml) was then added to the solution of liposome with adjuvant (425 ml) and stirred for 30 min at 60 C. resulting in a solution having a final B-OG concentration of (0.20%), which is far below the detergent's CMC (0.73%) (Dilution step). The resulting solution was then concentrated by ultrafiltration (same condition mentioned above) and the final vaccine volume set to 50 ml. The concentrated solution was dialysed by diafiltration, where a 10 volume exchange was performed with PBS pH 7.4.

(44) In a final step, the vaccine solution was sterile filtered through a 0.2 um Acetate cellulose filter (Minisart 16534). Each filter was used to sterile filter 5 ml of vaccine solution into 15 ml Falcon tubes. This last process step is executed in a sterile environment (laminar flow hood).

(45) Scalability of process L20 has been demonstrated by generating batches with different volumes (50 and 150 ml) and with identical biophysical and immunological properties.

(46) 1.4 Results

(47) Table 4 describes a batch of L16 process with normal MPLA concentration producing PaI 1-15 vaccine (ACI-24-091127-A). The batch was manufactured with process L16 (antigen and adjuvant added after liposome formation) as described in Example 1ACI-24-091127-A. Normal MPLA concentration means an identical MPLA incorporation as adopted for the process L15 described in the paragraph above.

(48) Table 5 describes a batch of L16 process with high MPLA concentration producing PaI 1-15 vaccine (ACI-24-091127-B). The batch was manufactured with process L16 (antigen and adjuvant added after liposome formation) as described in Example 1. High MPLA concentration means a MPLA load in the final vaccine formulation which is approximately 8 times higher than in the process L15 Such high MPLA yield is not obtainable with the cross-flow ethanol injection method (process D)

(49) The advantages of the L16/L15 processes for producing PaI 1-15 vaccine are evident when comparing the outcome of process D for producing PaI 1-15 vaccine (see Table 1 to 3, 11 and 12, respectively). As we can see in the tables, MPLA hydrolysis (reported by formation of congener B) was significantly reduced in process L15/L16 as compared to process D. Moreover, peptide distribution with process L15/L16 reported an increase of antigen exposure towards the outer aqueous surface as compared to process D (Table 14).

(50) FIG. 13 shows the IgG titers of mice immunized with a Tau vaccine generated either with process L15 or L20. As can be observed, identical titer yields are obtained for the two processes.

(51) Scalability of process L20 has been demonstrated by generating batches with different volumes (50 and 150 ml) and with identical biophysical and immunological properties.

(52) All processes that I have worked are theoretically stable, though we only have data for the L20 process

Example 2: Comparison of L15 Method Vs Thin Film Method Producing ACI-33 Vaccine

(53) Table 6 and 7 describe a batch of L15 process and thin film process (process A as described in WO2007/068411), respectively, for producing ACI-33 vaccines. In FIG. 2, the anti-Tau5-20 [pY18] IgG antibody titers in the plasma of C5BL/6 mice after receiving ACI-33 vaccines are shown, either manufactured with the thin film method (Process A) or with the process L15. Mice challenged with ACI-33 vaccines manufactured with process L15 gave higher antibody titers than mice challenged with a process A vaccine. This effect should be attributed to the L15 vaccine properties which not only display a higher peptide load with an exclusive peptide distribution but also to the fact that L15 process generates smaller liposomes (<200 nm) than those made by the thin-film method.

Example 3: Comparison of L15 Method Vs Thin Film Method Producing ACI-35 Vaccine

(54) Table 8 and 9 describe a batch of L15 process and thin film process (process A as described in WO2007/068411), respectively, for producing ACI-35 vaccine. In FIG. 3 the anti-TAU396-408 [pS396/pS404] IgG antibody titers in the plasma of C5BL/6 mice after receiving ACI-35 vaccines are shown, either manufactured with the thin film method or with the process L15. Mice challenged with ACI-35 vaccine manufactured by process L15 gave higher antibody titers than mice challenged with a process A vaccine. This effect should be attributed to the L15 vaccine properties which not only allow a higher peptide load with an exclusive peptide distribution but also to the fact that L15 process generates smaller liposomes (<200 nm) than the thin-film method.

Example 4: Comparison of L15 Method Vs Cross Flow Ethanol Injection (Process D) Method Producing T1 Vaccine

(55) FIG. 4 shows the anti-A IgG antibody titers in the plasma of C5BL/6 mice after receiving PaI 1-15 vaccines, either manufactured with the process D (ACI-24 process D #2) or with the process L15. Results highlight identical antibody titers for the two vaccines. However, L15 process only contains negligible amounts of MPLA hydrolysis products (e.g. MPLA congener B) as compared to the other method (process D) where MPLA hydrolysis occurs to a much vast extent in a non-controlled fashion. The immunogenicity of different MPLA congeners (e.g. congener A which corresponds to its non hydrolyzed form and congener B, which is one of congener A hydrolyzate products) are not know. A process which does not contain the MPLA hydrolysis products has the advantage of a high batch reproducibility and the vaccines produced by such a process show improved quality and stability.

Example 5: In-Vivo Comparison of Method L15 Vs L16 and Immune Reproducibility of Process L15

(56) FIG. 5 shows the anti-A IgG antibody titers in the plasma of C5BL/6 mice after receiving PaI 1-15 vaccines, either manufactured with the process D (ACI-24 process D #1), with the 3 independently generated L15 vaccines and one vaccine generated with the L16 process. Results show identical antibody titers for the 3 individual L15 vaccines highlighting in-vivo immunological reproducibility. At day 35 after immunization there were no major differences in the antibody titers between L15 vaccine and process D vaccine. However, MPLA was hydrolyzed to a much greater extent in the ethanol cross-flow injection method (process D) than compared to the L15 or L16 processes.

(57) The antibody titers obtained with the L16 process are slightly lower at day 35 when compared to L15 batches. This result may be due to an excess of MPLA on the external phospholipid bilayer as compared to the process L15. Different doses of MPLA should be tested on both methods in order to obtain a clearer response on effect of adjuvant concentration on the immune response. However, the fact that MPLA can be added after liposome formation offers the advantage of incorporating the adjuvant only when needed into stocked empty-liposomes. This approach may prevent MPLA hydrolysis during storage in a liposome formulation.

Example 6: In-Vivo Generation of Two Different Antibodies by Generating Liposomes with the L15 Process Containing Two Antigens (T8 and T9 Antigen on ACI-41 Vaccine)

(58) Table 10 describes a batch of L15 process for producing a vaccine containing a mixture of T8 and T9 peptide sequences. The differences in T8 and T9 peptide solubility and purity may affect the final antigen yield in the vaccine formulation. In FIGS. 6a and 6b it is shown that ACI-41 vaccine (containing two different antigensT8 and T9) can induce a specific antibody response for the two epitopes present on the same liposomes.

Example 7: In-Vivo Generation of Antibodies by Generating Liposomes with the L15 Process Containing Different Adjuvants than MPLA: Lipidated CpG Adjuvant (Vaccine ACI-17) or Pam2CSK4 (Vaccine ACI-18)

(59) Vaccines ACI-17 and ACI-18 manufactured by process L15 were prepared as previously described in Example 1.2 with the only difference in adjuvant selection. Adjuvants used in vaccines ACI-17 and ACI-18 were lipidated CpG and Pam2CSK4, respectively.

(60) FIG. 14 shows the anti-A IgG antibody titers in the plasma of C5BL/6 mice after receiving PaI 1-15 vaccines, manufactured with the process L15 where MPLA is replaced by lipidated CpG adjuvant (vaccine ACI-17) or by Pam2CSK4 (vaccine ACI-18). Results highlight the technology flexibility in loading the liposomes with adjuvants different from MPLA and still inducing an immune response which generates antibodies which bind to A.

Example 8: Determination of the Membrane Topology of PaI1-15 in ACI-24 Using a BCA Colorimetric Assay

(61) The Bicinchoninic acid protein quantification assay (BCA) was developed and tested for linearity, specificity and precision. Finally the assay was implemented to analyse the peptide topology in different batches of ACI-24 prepared by different processes. The BCA assay is based upon a two-step reaction in which Copper(II) is firstly reduced to Copper(I) in the presence of peptide under basic conditions (Biuret reaction). In a second step, Copper(I) chelates with the reagent Bicinchoninic acid to generate a purple colored complex that can be measured by Absorbance and which is proportional to the peptide content. Due to the charge of Copper(II) and Bicinchoninic acid, these reagents would not be expected to cross the liposome bilayer and so should quantify only peptide on the outer membrane. To quantify the total peptide content, the liposomes can be lysed in the presence of a detergent and then the quantification performed using the BCA reagent. The proportion of PaI1-15 on the outer surface can then be determined from the ratio of peptide on the outer surface and total peptide. As a control for specificity, liposomes lacking peptide (empty liposomes) were used. Likewise, in order to ensure that peptide on the outer surface could be quantified and that the bicinchoninic acid and copper would not traverse the liposome membrane, a control batch composed of a water soluble peptide Ac1-15 fully encapsulated within the interior of liposomes was tested.

(62) The standard BCA assay conditions were modified in order to optimize the signal/noise ratio as well as the reaction specificity. The parameters that were optimized include i) concentration of bicinchoninic acid and copper (II), ii) concentration of ACI-24, iii) reaction temperature and iv) reaction time (data not shown).

(63) 8.1 Peptide on Outer Liposome Surface

(64) Liposomes were diluted 2-fold with PBS and 240 L added to a 96-well flat-bottom transparent plate. 60 L of 4 concentrated BCA reagent (micro-BCA Protein Assay Kit, 1.88 mL reagent A, 1.80 mL reagent B, 256 L reagent C) was added and the samples mixed and left at RT in the absence of light for 90 min.

(65) 8.2 Total Peptide Content

(66) In order to lyse liposomes to quantitate total peptide content, liposomes were diluted 2-fold with SDS to give a final SDS concentration of 2.25% (v/v). 300 L of liposomes in SDS was then heated in a sealed plastic eppendorf at 70 C. for 2 h and then cooled to RT over 2 h. The efficiency of lysing could be followed by monitoring the Absorbance in the range 320-600 nm. 240 L of this transparent solution was then added to a 96-well flat-bottom transparent plate. 60 L of 4 concentrated BCA reagent was added and the sample mixed and left at RT in the absence of light for 90 min.

(67) 8.3 Absorbance Analysis

(68) Absorbance measurements were performed over the range 410-700 nm and the Abs at 562 nm was used to calculate the proportion of peptide on the outer liposome surface. Since liposomes scatter light due to their large size, this background absorbance is corrected by subtraction from the absorbance of liposomes measured with the BCA assay according to the following formula:
% peptide on outer membrane=(Abs liposomes+BCA)(Abs liposomes)/(Abs lysed liposomes+BCA)(Abs lysed liposomes)

(69) 8.4 Results

(70) 8.4.1 Linearity

(71) To determine the linear range of the assay, standards of the water soluble peptide Ac1-15 (Polypeptides, France) was analyzed, upon solubilzation in PBS, with the BCA assay in the range 6.25.fwdarw.100 M final peptide concentration. Good linearity was found over this range (R.sup.2>0.97) (FIG. 7). ACI-24 samples are analyzed at 2-fold dilution which means a theoretical peptide concentration of 60 M which is thus well within the linear range of the assay.

(72) 8.4.2 Specificity

(73) 8.4.2.1 Effect of SDS:

(74) In order to ensure that the presence of 2.25% SDS detergent would not interfere with the assay, standards of Ac1-15 peptide were prepared in SDS as for liposome samples. As can be seen in FIG. 7, Ac1-15 solubilized in SDS gave a similar standard curve compared with that solubilized in PBS.

(75) 8.4.2.2 Effect of ACI-24 Liposome Samples:

(76) Absorbance spectra of an ACI-24 batch (process L15) are shown in FIG. 8. Strong absorbance is seen at 562 nm characteristic of the BCA-Cu(I) complex, both for ACI-24 treated with BCA reagents either in the presence of PBS or SDS. No peaks were observed for liposomes in PBS or SDS without treatment with BCA reagents. As expected, liposomes in PBS alone give rise to background absorbance at 562 nm whereas liposomes lysed with SDS to give micelles show only minor absorbance over the range 410.fwdarw.700 nm. When the background absorbance is corrected, the absorbance spectra of ACI-24 in PBS or SDS are similar but differ only in signal intensity at close to 562 nm (FIG. 9).

(77) 8.4.2.3 Effect of Liposome Matrix:

(78) In order to determine whether the Liposome matrix could interfere with the BCA assay, a batch of liposomes identical to ACI-24 but lacking peptide (ACI-24E-100316) was analyzed. As can be seen in FIG. 10, no peak is observed at 562 nm demonstrating that the abs peak at 562 nm observed for ACI-24 liposomes is due to the presence of peptide.

(79) 8.4.2.4 Specificity for Peptide Only on Outer Membrane:

(80) In order to test whether the BCA assay performed with liposomes diluted with PBS specifically reacts only with peptide present on the outer liposome membrane, the assay was performed with a batch of liposomes containing encapsulated water-soluble peptide Ac1-15 (ACI-16). As can be seen in FIG. 11, essentially no peak is observed at 562 nm, thus confirming that the BCA reaction occurs only for peptide exposed on the bilayer outer surface.

(81) 8.4.3 Precision

(82) To assess the assay precision triplicate analyses were performed a batch of ACI-24 prepared with process L15 (ACI-24-100316-A). The results show that the absorbance readings in both PBS and SDS have coefficient of variations (CV's) of 1.77% and 0.57% respectively (Table 13).

(83) 8.4.4 Batch Analyses

(84) Comparison of Liposomes Prepared with Different Processes

(85) Different batches of ACI-24 were analyzed in order to determine the effect of different liposome production processes upon the membrane topology of the peptide in the liposomes. The absorbance spectra of selected batches are shown in FIG. 12 and summarized in Table 14.

(86) The results provided in Table 14 demonstrate the process flexibility in terms of volumes, adjuvants (more than one adjuvant), antigens (more than one antigen), lipid compositions
that may be used in the process of the invention.

(87) Implementation of the assay for batch analyses revealed that liposomes prepared by different processes have different proportions of peptide present on the outer membrane surface. In particular, liposomes prepared with process L15 were found to display close to 30% more peptide on the outer surface than those prepared with the thin-film process A.

(88) While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. It will be understood that changes and modifications may be made by those of ordinary skill within the scope and spirit of the following claims. In particular, the present invention covers further embodiments with any combination of features from different embodiments described above and below.

(89) The invention also covers all further features shown in the Figures individually although they may not have been described in the afore or following description.

(90) Tables

(91) TABLE-US-00003 TABLE 1 ACI-24-100316-A Characteristic Test Method Results Apperance Visual White, milky inspection suspension Content Pal1-15 HPLC 424 ug/ml Content membrane HPLC 98% bound Pal1-15 Content MPLA HPLC 101 ug/ml Congener A HPLC 99 ug/ml Congener B HPLC 2 ug/ml Size DLS 110 nm Polydispersity DLS 0.25

(92) TABLE-US-00004 TABLE 2 ACI-24-100316-B Characteristic Test Method Results Apperance Visual White, milky inspection suspension Content Pal1-15 HPLC 482 ug/ml Content membrane HPLC 110% bound Pal1-15 Content MPLA HPLC 103 ug/ml Congener A HPLC 100 ug/ml Congener B HPLC 3 ug/ml Size DLS 110 nm Polydispersity DLS 0.25

(93) TABLE-US-00005 TABLE 3 ACI-24-100316-C Characteristic Test Method Results Apperance Visual White, milky inspection suspension Content Pal1-15 HPLC 406 ug/ml Content membrane HPLC 93% bound Pal1-15 Content MPLA HPLC 102 ug/ml Congener A HPLC 99 ug/ml Congener B HPLC 3 ug/ml Size DLS 110 nm Polydispersity DLS 0.25

(94) TABLE-US-00006 TABLE 4 ACI-24-091127-A Characteristic Test Method Results Apperance Visual White, milky inspection suspension Content Pal1-15 HPLC 647 ug/ml Content membrane HPLC 73% bound Pal1-15 Content MPLA HPLC 250 ug/ml Congener A HPLC 214 ug/ml Congener B HPLC 36 ug/ml Size DLS 109 nm Polydispersity DLS 0.9

(95) TABLE-US-00007 TABLE 5 ACI-24-091127-B Characteristic Test Method Results Apperance Visual White, milky inspection suspension Content Pal1-15 HPLC 543 ug/ml Content membrane HPLC 88% bound Pal1-15 Content MPLA HPLC 884 ug/ml Congener A HPLC 818 ug/ml Congener B HPLC 66 ug/ml Size DLS 117 nm Polydispersity DLS 0.30

(96) TABLE-US-00008 TABLE 6 ACI-33-091127 Characteristic Test Method Results Apperance Visual White, milky inspection suspension Content T1 HPLC 348 ug/ml Content membrane HPLC 87% bound T1 Content MPLA HPLC 96 ug/ml Congener A HPLC 81 ug/ml Congener B HPLC 15 ug/ml Size DLS 106 nm Polydispersity DLS 0.18

(97) TABLE-US-00009 TABLE 7 ACI-33-091808-A Characteristic Test Method Results Apperance Visual White, milky inspection suspension Content T1 HPLC 63 ug/ml Content membrane HPLC ND bound T1 Content MPLA HPLC 79 ug/ml Congener A HPLC 79 ug/ml Congener B HPLC 0 ug/ml Size DLS ND Polydispersity DLS ND

(98) TABLE-US-00010 TABLE 8 ACI-35-091127 Characteristic Test Method Results Apperance Visual White, milky inspection suspension Content T3 HPLC 321 ug/ml Content membrane HPLC 109% bound T3 Content MPLA HPLC 101 ug/ml Congener A HPLC 96 ug/ml Congener B HPLC 5 ug/ml Size DLS 102 nm Polydispersity DLS 0.25

(99) TABLE-US-00011 TABLE 9 ACI-35-0910820-A Characteristic Test Method Results Apperance Visual White, milky inspection suspension Content T3 HPLC 61 ug/ml Content membrane HPLC ND bound T3 Content MPLA HPLC 141 ug/ml Congener A HPLC 141 ug/ml Congener B HPLC 0 ug/ml Size DLS ND Polydispersity DLS ND

(100) TABLE-US-00012 TABLE 10 ACI-41-100531 Characteristic Test Method Results Apperance Visual White, milky inspection suspension Content T8 HPLC 362 ug/ml Content T9 HPLC 189 ug/ml Content MPLA HPLC 93 ug/ml Congener A HPLC 88 ug/ml Congener B HPLC 5 ug/ml Size DLS 79 nm Polydispersity DLS 0.25

(101) TABLE-US-00013 TABLE 11 ACI-24 process D#1 Characteristic Test Method Results Apperance Visual White, milky inspection suspension Content Pal1-15 HPLC 388 ug/ml Content membrane HPLC ND bound Pal1-15 Content MPLA HPLC 58 ug/ml Congener A HPLC 12 ug/ml Congener B HPLC 46 ug/ml Size DLS 115 nm Polydispersity DLS 0.14

(102) TABLE-US-00014 TABLE 12 ACI-24 process D#2 Characteristic Test Method Results Apperance Visual White, milky inspection suspension Content Pal1-15 HPLC 375 ug/ml Content membrane HPLC ND bound Pal1-15 Content MPLA HPLC 52 ug/ml Congener A HPLC 11 ug/ml Congener B HPLC 41 ug/ml Size DLS 105 nm Polydispersity DLS 0.15

(103) TABLE-US-00015 TABLE 13 Sample CV conditions Abs 1 Abs 2 Abs 3 Average S.D. (%) SDS with BCA 1.174 1.152 1.157 SDS without 0.052 0.042 0.045 BCA Difference 1.122 1.110 1.112 1.115 0.006 0.57 PBS with BCA 1.315 1.313 1.281 PBS without 0.464 0.454 0.451 BCA Difference 0.852 0.859 0.830 0.847 0.015 1.77

(104) TABLE-US-00016 TABLE 14 Peptide added Relative Adjuvant before or Peptide Antigen added after on outer added after after Production liposome Batch membrane Size MPLA Process liposome liposome Process formation name (%) (nm) Hydrlosis Filterability Scalable Flexibility formation formation A (thin-film) Before ACI-24- 54 >500 nm No No No Low No No as described 090813-A in WO2007/068411 D (cross-flow Before ACI240108-B 62 96 Yes Yes Yes Low No No ethanol ACI240709-A 58 115 Yes Yes Yes Low No No injection) ACI241008-A 67 97 Yes Yes Yes Low No No ACI240908-A 64 105 Yes Yes Yes Low No No L15 After ACI-24- 81 116 No Yes Yes High Yes No 091016-B ACI-24- 81 110 No Yes Yes High Yes No 100316-A L16 After ACI-24- 82 109 No Yes Yes High Yes Yes 091127-A ACI-24- 84 117 No Yes Yes High Yes Yes 091127-B ACI-24- 72 113 No Yes Yes High Yes Yes 100317-C L20 After ACI001J 84 101 No Yes Yes High Yes No Process Flexibility means that the process can be used for various volumes, adjuvants (more than one adjuvant), antigens (more than one antigen), lipid compositions.

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