FORMULATION OF A PEPTIDE VACCINE
20230233658 · 2023-07-27
Assignee
Inventors
Cpc classification
A61K9/0019
HUMAN NECESSITIES
C12N2710/20034
CHEMISTRY; METALLURGY
A61K47/14
HUMAN NECESSITIES
A61K47/10
HUMAN NECESSITIES
A61K9/1075
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
A61K47/10
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The invention relates to a novel reconstitution composition, a pharmaceutical composition and kit of parts comprising said reconstitution composition. The invention further relates to a method of treatment using said pharmaceutical composition and/or the pharmaceutical composition for use as a medicament. Also provided is a method for reconstituting dried peptides and a method for preparing a pharmaceutical composition using the reconstitution composition of the invention.
Claims
1.-20. (canceled)
21. A combination comprising: (a) a first vial comprising: (i) 5 peptides consisting of the sequences set forth in SEQ ID NOs: 1-5; or (ii) 6 peptides consisting of the sequences set forth in SEQ ID NOs: 1-6; or (iii) 7 peptides consisting of the sequences set forth in SEQ ID NOs: 7-13.
22. The combination according to claim 21, further comprising a second vial comprising an adjuvant.
23. The combination according to claim 22, wherein the adjuvant is an oil-based adjuvant, or wherein the adjuvant is selected from a mineral or non-mineral oil-based adjuvant.
24. The combination according to claim 22, wherein the adjuvant is an immune-stimulating adjuvant.
25. The combination according to claim 21, wherein the mix of peptides is lyophilized.
26. The combination according to claim 21, wherein the mix of peptides consists of the 5 different peptides consisting of the sequences set forth in SEQ ID NOs: 1-5 or wherein the mix of peptides consists of the 7 different peptides consisting of the sequences set forth in SEQ ID NOs: 7-13.
27. The combination according to claim 23, wherein the oil-based adjuvant is selected from bio-based oil adjuvants, adjuvants based on vegetable oil or fish oil, squalene-based adjuvant, MF59, Syntex Adjuvant Formulation, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, adjuvants based on peanut oil, Adjuvant 65, Lipovant, ASO4, Montanide adjuvants, adjuvants based on purified squalene and squalene emulsified with highly purified mannide mono-oleate, and a mixture of Drakeol VR and mannide monooleate.
28. The combination according to claim 21, further comprising: (c) a vial comprising a composition for reconstituting dried peptides, wherein the composition comprises or consists of 60-80% v/v aqueous solution comprising an organic acid, 5-10% v/v propylene glycol (CAS no. 57-55-6), 10-20% v/v lower alcohol, and 5-10% v/v non-ionic hydrophilic surfactant.
29. The combination according to claim 28, wherein the organic acid is citric acid.
30. The combination according to claim 28, wherein the lower alcohol is ethanol.
31. The combination according to claim 28, wherein the non-ionic hydrophilic surfactant is ethoxylated castor oil or: a. is a mono-, di or triglyceride, and/or b. has a hydrophilic-lipophilic balance (HLB) value between 9 and 14.
32. The combination according to claim 28, wherein the composition for reconstituting dried peptides comprises or consists of 75% v/v aqueous solution comprising 0.1M citric acid, 6.25% v/v propylene glycol (CAS no. 57-55-6), 12.5% v/v ethanol, and 6.25% v/v polyoxyethyleneglyceroltriricinoleate 35 (CAS no. 61791-12-6).
33. A pharmaceutical composition comprising: (i) 5 peptides consisting of the sequences set forth in SEQ ID NOs: 1-5; or (ii) 6 peptides consisting of the sequences set forth in SEQ ID NOs: 1-6; or (iii) 7 peptides consisting of the sequences set forth in SEQ ID NOs: 7-13.
34. The pharmaceutical composition according to claim 33, further comprising an adjuvant.
35. The pharmaceutical composition according to claim 34, wherein the adjuvant is an immune-stimulating adjuvant.
36. The pharmaceutical composition according to claim 34, wherein the adjuvant is an oil-based adjuvant or wherein the adjuvant is selected from a mineral or non-mineral oil-based adjuvant.
37. The pharmaceutical composition according to claim 36, wherein the oil-based adjuvant is selected from bio-based oil adjuvants, adjuvants based on vegetable oil or fish oil, squalene-based adjuvant, MF59, Syntex Adjuvant Formulation, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, adjuvants based on peanut oil, Adjuvant 65, Lipovant, ASO4, Montanide adjuvants, adjuvants based on purified squalene and squalene emulsified with highly purified mannide mono-oleate, and a mixture of Drakeol VR and mannide monooleate.
38. The pharmaceutical composition according to claim 34, wherein the composition comprises or consists of 1-2 mg/mL peptides in 40-60% v/v of the reconstitution composition and 40-60% v/v of Montanide ISA 51VG.
39. The pharmaceutical composition according to claim 33, wherein the mix of peptides is chemically stable for at least 2 hours at room temperature.
40. A method for making a stable pharmaceutical composition by selecting peptides selected from: (i) 5 peptides consisting of the sequences set forth in SEQ ID NOs: 1-5; or (ii) 6 peptides consisting of the sequences set forth in SEQ ID NOs: 1-6; or (iii) 7 peptides consisting of the sequences set forth in SEQ ID NOs: 7-13 and reconstitution the peptides in reconstitution solution comprising 60-80% v/v aqueous solution comprising an organic acid, 5-10% v/v propylene glycol, 10-20% v/v lower alcohol, and 5-10% v/v non-ionic hydrophilic surfactant.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
Example 1
Introduction
[0142] The aim of this study was to find a suitable reconstitution method for a multipeptide HPV vaccine product involving dissolution of the peptide Drug Products HPV-DP-6P and HPV-DP-7P, followed by emulsification with Montanide ISA51VG. Previous studies have shown that in DMSO/WFI formulations, peptides containing one or more cysteine residues have a strong tendency to form disulfides. To improve the chemical stability of the Drug Products and prevent disulfide formation of the peptides, a new DMSO-free reconstitution solution was developed for reconstitution of both Drug Products. This new reconstitution solution should be able to dissolve the Drug Product and result in a stable emulsion with Montanide ISA51VG. Disulfide-formation should be minimal.
[0143] The study consists of four levels of analysis: [0144] 1. Screening for a suitable solvent combination to reconstitute the Drug Products, monitoring dissolution of the peptides by visual inspection. [0145] 2. Monitoring of the emulsion stability of the Drug Product emulsion with Montanide. Stability is assessed by visual inspection and by analysis of particle size of the emulsion droplets. [0146] 3. Analysis of the chemical stability of the Drug Product after reconstitution in solvents that were successful on level 1 and 2. [0147] 4. Analysis of the chemical stability of the Drug Product after reconstitution and emulsification, using solvents that were successful in level 1, 2 and 3. For this purpose, the peptides are dissolved, emulsified with Montanide ISA 51 VG, followed by extraction from the emulsion and analysis of the peptide composition.
Materials
[0148] The following lyophilized peptide compositions were used: DP-5P comprising peptides represented herein by SEQ ID NO: 1-5 admixed at equal net weights of 0.40 mg of each peptide per vial (total amount of protein per vial being 2.00 mg) and 0.56 mg TFA per vial; DP-6P comprising peptides represented herein by SEQ ID NO: 1-6 admixed at equal net weights 0.40 mg of each peptide per vial (total amount of protein per vial being 2.40 mg) and 0.67 mg TFA per vial; and DP-7P comprising peptides represented herein by SEQ ID NO: 7-13 admixed at equal net weights of 0.40 mg of each peptide per vial (total amount of protein per vial being 2.80 mg) and 0.96 mg TFA per vial.
[0149] The following chemicals were used: Cremophor EL, (Sigma Aldrich, Kolliphor EL, C5135); Propylene Glycol or PG (≥99.5%, Sigma Aldrich, W294004) Ethanol or EtOH (Absolute, VWR Emprove® Ph Eur, BP, USP. Article #1.00986.1000); Citric acid or CA (≥99%, Sigma Aldrich C1909); MilliQ water (from EQP-063); Sterile Montanide ISA 51VG (SEPPIC, batch #14V011).
[0150] The following equipment was used: Syringe extrusion devices (Discofix-3 T-connector, B. Braun); DMSO-resistant syringes (2 mL NORM-JECT Luer Lock, Henke Sass Wolf); Waters UPLC/MS system; Malvern Mastersizer 2000; Protein Simple MFI 5200 flowcell.
Methods
Dissolution
[0151] Reconstitution composition was prepared by mixing the organic and aqueous solvents before adding them to the lyophilized Drug Product. 1 mL of various reconstitution compositions was added to the Drug Product and the mixture was allowed to stand for 5 minutes, while swirling the solution several times. Physical stability was assessed by visual inspection. Chemical stability was assessed using UPLC/MS (see below under Chemical stability of the Drug Product solution).
Emulsification with Montanide
[0152] Solvent combinations resulting in a visually clear Drug Product solution were used in emulsification experiments with Montanide ISA51 VG. Unless stated otherwise, reconstitution and emulsification was performed according to the protocol in Table 1. Where indicated, mixing of the contents of syringe A and B was performed differently. These adaptations of the procedure in Table 3 are indicated in the results section in Table 4 and Table 5.
TABLE-US-00002 TABLE 1 Reconstitution and emulsification of drug product (DP). Step Description 1 At least 10 minutes and maximum 30 minutes before start formulation, thaw at room temperature 1 vial with DP, lyophilized powder for injection. Record time of removal from the freezer (hh;min). 2 Collect 1 mL reconstitution composition in a 2 mL syringe. 3 Record time of starting the reconstitution (hh;min). 4 Add the content of the syringe containing sterile reconstitution composition (1 mL) to the DP vial. Do not swirl the vial. Remove the syringe from the vial. 5 Allow the mixture to stand for 2 minutes at RT, followed by gentle swirling for 3 minutes. If the content of the vial is not completely dissolved, vortex for 30 seconds. 6 Collect the contents of the vial (1.0 mL) in a new syringe (syringe A). 7 Collect 1.0 mL Montanide ISA 51 VG in a third 2 mL syringe (syringe B). 8 Remove one of the white caps of the T- connector and firmly attach the syringe containing the peptide solution in reconstitution composition (1.0 mL) to the connector (Syringe A). 9 Remove the second white cap of the T- connector and attach the syringe containing 1.0 mL Montanide ISA 51 (Syringe B) to the connector. 10 Turn the switch-key and push the content of syringe A first slowly into syringe B and then from syringe B to A. This is 1 cycle. Start the stopwatch. Repeat the cycle in total 50 times 40-50 seconds. Record number of seconds (to be documented by second operator). 11 Collect the vaccine emulsion in one syringe. Remove the syringe from the T- connector and place a clean needle on the syringe.
Laser Diffraction Experiments for Testing Emulsion Stability
[0153] Emulsion stability was monitored both by visual inspection and by analysis of the particle size distribution using a Malvern Mastersizer 2000.
[0154] For particle size analysis, dilution of the emulsion was performed either with water or with a 0.01 M citric acid in water solution to obtain the desired level of obscuration. Montanide was admixed with the reconstitution composition comprising reconstituted DP using a stirrer at a speed of 1750 rpm and a refractive index of 1.46 were applied. Particle size distribution was expressed in D(0.5) and D(0.9) for a volume-based distribution.
Micro Flow Imaging (MFI) for Testing Emulsion Stability
[0155] As a second technique for particle size analysis for assessing emulsion stability, Micro Flow Imaging was used. Prior to analysis, a dilution of the emulsion was prepared by adding one droplet of emulsion to 10 mL 0.01M aqueous citric acid solution and mixing until homogeneous, followed by 1:100 dilution of this solution in water. Samples were measured in a purge volume of 0.20 mL for the duration of 0.68 minutes or per 1 million particles in one single run. The results are expressed in Equivalent Circle Diameter (ECD).
Chemical Stability of the Drug Product Solution
[0156] For samples showing complete dissolution and an emulsion stability of >2 hours, the chemical stability of the Drug Product solutions (without additional dilution) was monitored with UPLC/MS on a Waters Acquity UPLC system coupled to a Waters TQD mass spectrometer using a Waters Acquity column (type: BEH130, C18, 1.7 μm, 2.1×150 mm). Data processing was performed with Masslynx 4.1 software. UV-detection was performed at 220 nm and the mobile phase was 0.05% TFA and 1% ACN in water (buffer A) and 0.05% TFA in ACN (buffer B) at a flowrate of 0.3 mL/min. The column temperature was 65° C. and the autosampler temperature was 5° C. An injection volume of 5 μL was used, and the gradient profile of Table 2 was applied.
[0157] UV-detection was performed during the full length of the gradient, and mass spectrometric analysis was performed from 2-30 min in the positive mode.
[0158] For analysis of chemical stability of the Drug Product solutions, samples were analyzed at various time points, at least up to 2 hours after dissolution.
TABLE-US-00003 TABLE 2 Gradient profile for UPLC/MS. Time Eluent A Eluent B (min) (%) (%) 0 87 13 0.5 87 13 5.5 79.5 20.5 17.0 68 32 22.8 45 55 28.5 45 55 28.6 20 80 30.0 20 80 30.1 87 13 33.0 87 13
In-Use Chemical Stability of HPV-DP-6P and HPV-DP-7P Vaccine Emulsions
[0159] For samples showing complete dissolution, an emulsion stability of >2 hours, and a chemical stability of the Drug Product solutions (without additional dilution) of >2 hours, the in-use chemical stability of the vaccine emulsions with Montanide ISA 51 VG was monitored with UPLC/MS. For analysis of chemical stability of the reconstituted and emulsified Drug Products, samples were analyzed at various time points, at least up to 2 hours after dissolution. UPLC/MS analysis was performed according to the method describe above for chemical stability of the Drug Product solution, using an extra sample preparation step for extraction of the peptides from the vaccine emulsion. For sample preparation of emulsified products, the following steps were applied: [0160] Take 300 μL Reconstitution Solution and add this to a 15 mL Greiner tube [0161] Add 100 μL heptane [0162] Add 200 μL of the Drug Product emulsion. Pipet the solution up and down three times. [0163] Vortex the mixture for 30 seconds [0164] Centrifuge the mixture for 5 minutes at 4400 rpm to obtain a two-phase system [0165] With a 20-200 μL pipette, take a 100 μL sample from the bottom layer and transfer to a total recovery UPLC vial. [0166] Analyze with UPLC/UV/MS according to the method described for chemical stability of the Drug Product solutions.
Results
Solvent Screening for Reconstitution and Emulsification
[0167] Solvents were screened to define a reconstitution composition comprising both an aqueous and organic fraction that is suitable for reconstituting lyophilized peptides and forming a chemically and physically stable emulsion with Montanide. All experiments below were performed with DP-6P. The experiments were verified using DP-5P and DP-7P, but as data were highly comparable, only the data on DP-6P are shown here. Physical stability of the reconstituted proteins and emulsion in this screen was assessed by visual inspection.
[0168] As organic fraction, a wide variety of organic solvents was tested. The only single organic solvent capable of completely dissolving DP-6P when admixed with WFI (water for injection) was NMP (Table 3). However, no stable emulsion with Montanide could be obtained when using NMP/WFI as reconstitution composition. The use of saline instead of WFI slightly improved the emulsion stability, but still no emulsions with a stability of ≥2 h could be obtained in a reproducible manner.
TABLE-US-00004 TABLE 3 Solvent screening for dissolution of DP-6P. Peptides Aqueous Organic 1 solubility Emulsion stability 600 μL WFI 400 μL Particles NA Glycerol 600 μL WFI 400 μL Clear viscous NA PG solution 600 μL WFI 400 μL Particles NA EtOH WFI 100-20% Particles NA DMF 800 μL WFI 200 μL Clear homogeneous < 1 h NMP solution 800 μL WFI, 200 μL Clear homogeneous < 2 h 0.9% NaCl NMP solution
Organic Solvent Mixtures in Reconstitution
[0169] No single organic solvent was identified that in combination with WFI resulted in complete dissolution of DP-6P. Therefore, combinations of propylene glycol and other solvents were screened as organic fraction in the reconstitution composition. Physical stability was assessed by visual inspection. Chemical stability was assessed using UPLC/MS.
[0170] Although still no complete dissolution of DP-6P was observed, the most optional solvent combination identified for dissolution of DP-6P was a mixture of ethanol, propylene glycol and Cremophor EL as emulsifier with WFI (
[0171] To further improve the dissolution process while limiting disulfide formation, the effect of adding several antioxidants and reducing agents to the solvent mixture (mixture of ethanol, propylene glycol, Cremophor EL and WFI) and the peptide solution was assessed. Chemical stability was analyzed with UPLC/MS to monitor the extent of disulfide formation. Addition of DTT (35 molar equivalents compared to peptide) or ascorbic acid (0.1-1% solution in WFI) did not result in a reduction of disulfide formation, whereas the addition of a 0.05-0.1 M aqueous citric acid solution to the solvent mixture resulted in both improved dissolution of DP-6P and limited disulfide formation of area % values of ≤1% per disulfide two hours after dissolution of the Drug Product. Citrate buffer at pH3 and a concentration of 0.05-0.1 M could not be used for emulsification because of poor peptide dissolution (data not shown).
Testing Reconstitution Compositions after Reconstitution and after Subsequent Emulsification
[0172] Cremophor EL as emulsifier is less preferred in vaccine formulations because of reported side effects at higher dosages. However, the dissolving and emulsifying properties of Tween 80, cyclodextrins, and Triton X as alternatives for Cremophor EL, were inadequate (data not shown). Upon visual inspection, promising results were obtained with a combination of propylene glycol, ethanol, citric acid in WFI and 2% Tween20. The results of emulsification experiments as summarized in Table 4 show that emulsions comprising propylene glycol and ethanol in combination with either Cremophor EL or Tween 20 result in most stable emulsions. However, it appeared that the chemical stability in solution of both DP-6P and DP-7P was significantly worse in the presence of Tween20 instead of Cremophor EL, i.e. with area % values of over 5% per disulfide two hours after dissolution of the Drug Product (see
[0173] Taken together, from the data presented in Table 4 and
[0174] To demonstrate that the results obtained for chemical stability of Drug Product in solution can be translated to the in-use chemical stability of the Drug Product in the vaccine emulsion, the in-use stability of DP-6P and DP-7P vaccine emulsions was studied and results are presented in
TABLE-US-00005 TABLE 4 Solvent screening with premixed organic and aqueous solvents (1 mL). Physical Citric acid Peptides emulsion solution Organic 1 Organic 2 Organic 3 solubility stability 0.1M, PG (3) EtOH (2) Cremophor + − 800 μL EL (1) 0.1M, PG (1) 0 Cremophor − − 800 μL EL (1) 0.1M, PG (2) EtOH (1) Cremophor + − 800 μL EL (1) 0.1M, PG (3) EtOH (3) Cremophor − NA 800 μL EL (2) 0.1M, PG (1) EtOH (1) Cremophor + − 800 μL EL (2) 0.1M, PG (2) EtOH (1) Cremophor −+ − 775 μL EL (1) 0.1M, PG (3) EtOH (2) Cremophor −+ − 750 μL EL (1) 0.1M, PG (1) EtOH (2) Cremophor −+ + 750 μL EL (1) 0.1M, 0 EtOH (4) Cremophor −+ − 750 μL EL (1) 0.1M, PG (3) EtOH (2) Cremophor − − 700 μL EL (1) 0.1M, PG (1) EtOH (2) Tween 20 −+ + 800 μL (1) 0.1M, PG (1) EtOH (2) Tween 20 −+ NA 750 μL (1) 0.1M, PG (1) EtOH (2) Tween 20 − NA 600 μL (1) 0.1M, PG (1) EtOH (2) Tween 20, −+ + 800 μL 50% aq (1) 0.1M, PG (1) EtOH (2) Tween 20, −+ + 800 μL 25% aq (1) 0.1M, PG (1) EtOH (2) Triton X − NA 750 μL (1) 0.1M, PG (1) EtOH (2) Triton X − NA 600 μL (1) 0.05M, PG (1) EtOH (1) Cremophor + − 800 μL EL (2) 0.05M, PG (2) EtOH (1) Cremophor −+ − 775 μL EL (1) 0.05M, PG (2) EtOH (1) Cremophor −+ + 750 μL EL (1) 0.05M, PG (2) EtOH (1) Cremophor − ++ 700 μL EL (1)
Fine-Tuning for Robustness in Emulsification
Peptide Solubility and Emulsion Stability
[0175] A subsequent series of experiments was performed in which the ratio of PG/EtOH/Cremophor EL was varied, two different concentrations of citric acid solution were tested, different emulsification methods were applied and the ratio of organic vs. aqueous components of the mixture was varied. In general, 1 mL reconstitution composition was prepared by mixing the organic and aqueous solvents before adding them to the lyophilized Drug Product. Subsequently, an emulsion was prepared by adding 1 mL Montanide to the 1 mL of aqueous peptide solution using different mixing steps and/or connectors as indicated in Table 5 and 6.
TABLE-US-00006 TABLE 5 Variation in emulsification method using different reconstitution compositions: Buffer Peptides Emulsion Emulsification method composition solubility stability 20 slow* cycles and A + − 80 fast* cycles 40 cycles in 40 sec A + − 20 slow cycles and B + − 80 fast cycles 10 slow cycles and B + − 40 fast cycles 20 slow cycles and C +− + 80 fast cycles 10 slow cycles and C +− +− 40 fast cycles 20 slow cycles and C +− + 40 fast cycles 40 fast cycles C +− + 40 fast cycles D +− + 20 slow cycles and E − ++ 40 fast cycles 40 fast cycles E − ++ *Slow cycles: 2 seconds per cycle. Fast cycles: 1 second per cycle. A = 800 μL 0.05M citric acid and 200 μL PG/EtOH/Cremophor EL (1:1:2); B = 800 μL 0.05M citric acid and 200 μL PG/EtOH/Cremophor EL (2:1:1); C = 750 μL 0.1M citric acid and 250 μL PG/EtOH/Cremophor EL (2:1:1); D = 750 μL 0.1M citric acid and 250 μL PG/EtOH/Cremophor EL (1:2:1); and, E = 700 μL 0.1M citric acid and 300 μL PG/EtOH Cremophor EL (2:1:1).
Peptide Emulsion Stability in More Detail: PSD Analysis by Laser Diffraction
[0176] For five different reconstitution compositions, the effect of different emulsification methods on particle size was analysed with laser diffraction, using a Malvern Mastersizer 2000. For all samples, 1 mL reconstitution composition was prepared by mixing the organic and aqueous solvents before adding them to the lyophilized Drug Product. Subsequently, an emulsion was prepared by adding 1 mL Montanide to the 1 mL of aqueous peptide solution. Mixing of the organic and aqueous phases was performed in three different ways: [0177] Using the T-connector process and performing mixing cycles as indicated in Table 6; [0178] Using an I-connector and performing mixing cycles as indicated in Table 6; or, [0179] Adding 1 mL Montanide to the vial containing the peptide solution in reconstitution composition, and vortexing the mixture during 30 seconds.
[0180] A summary of the results is presented in Table 6. Approximate values for D(0.5) are given (volume based distribution).
TABLE-US-00007 TABLE 6 Emulsification of peptide formulations characteristics and average D(0.5) values using different reconstitution compositions: Reconstitutio Emulsification composition process Solubility D(0.5) Stability A T-connector, 20 slow − 3 μm ≥3 h and 80 fast cycles A Vortex 30 seconds − 11 μm 3 h B T-connector, 20 slow +− 5-7 μm* ≥3 h and 80 fast cycles B T-connector, +− 9 μm ≥3 h 40 fast cycles B I-connector, 10 slow +− 11 μm ≥2 h and 40 fast cycles C T-connector, +− 4 μm ≥2 h 40 fast cycles D T-connector, +− 11 μm 1 h 40 fast cycles E T-connector, 20 slow + 11 μm 1 h and 80 fast cycles E I-connector, 20 slow + 12 μm 1 h and 80 fast cycles *Variation in PSD was observed for analysis diluted in WFI or diluted in 0.01M citric acid solution A = 600 μL 0.1M citric acid and 400 μL PG/EtOH/Cremophor EL (5:4:2); B = 750 μL 0.1M citric acid and 250 μL PG/EtOH/Cremophor EL (2:1:1); C = 750 μL 0.1M citric acid and 250 μL PG/EtOH/Cremophor EL (1:2:1); D = 775 μL 0.1M citric acid and 225 μL PG/EtOH/Cremophor EL (2:1:1); and, E = 800 μL 0.1M citric acid and 200 μL PG/EtOH/Cremophor EL (2:1:1).
[0181] Both from Table 5 and Table 6, it appears that no difference in emulsion stability was observed between the different mixing methods and/or different types of connectors used. However, vortexing the mixture instead of using a connector resulted in emulsions with a much larger particle size, which is less favorable for stability. In general emulsions with a smaller particle size are more stable.
[0182] Further, emulsion stability was improved by increasing the percentage of the organic fraction (mixture) in the total volume of reconstitution composition. However, the highest volumes of organic content tested here (300-400 μL) resulted in decreased solubility of the Drug Product. Therefore, the optimum of organic content was between 200 and 300 μL per mL (about 250 μL) reconstitution composition. In addition, variation in the concentration of the citric acid (0.05 or 0.1M) solution did not seem to affect the emulsion stability, while slightly better dissolution of DP-6P was obtained when a 0.1 M citric acid solution was used.
Particle Size Analysis Using Micro Flow Imaging
[0183] To study the effect of citric acid concentration on emulsion stability and particle size of the emulsion in more detail, additional particle size analysis experiments were compared using the solvent that resulted in the smallest particle size after emulsification with 1 mL Montanide, i.e. a reconstitution composition with an organic- to aqueous-phase ratio of 1:3, wherein the organic phase contains PG, EtOH and Cremophor EL in a ratio of PG to EtOH to Cremophor EL of 1:2:1 (Table 6). Direct comparison experiments were performed, wherein the molar amount of citric acid in the aqueous phase was varied (0.05 and 0.1M citric acid, i.e. an end concentration of citric acid in reconstitution composition of 0.038 and 0.075M citric acid, respectively). DP-6P was dissolved in 1 mL of such reconstitution composition, followed by emulsification with 1 mL Montanide using a T-connector process and performing 50 fast mixing cycles. In these experiments, both dissolution of the Drug Product and particle size and emulsion stability were analyzed. As a read-out, Micro Flow Imaging (MFI) was performed using an MFI 5200 in order to visualize the particles with a camera so that irregularities can be studied visually. The PSD-comparison of 0.05 and 0.1M citric acid of citric acid reconstitution composition is shown in
[0184] As can be seen in
Application of Preferred Reconstitution Solvent and Emulsification Method on DP-6P and DP-7P
[0185] Since 750 μL citric acid solution+250 μL PG/EtOH/Cremophor EL 1:2:1 was shown to give robust PSD results for DP-6P emulsions, and the use of 0.1M citric acid resulted in the best dissolution of the Drug Product, this solvent combination was tested extensively for the preparation of DP-6P and DP-7P emulsions.
[0186] DP-6P and DP-7P emulsions were prepared according to the instructions in Table 1. Briefly, 1 mL of reconstitution composition (750 μL 0.1M citric acid+250 μL PG/EtOH/Cremophor EL 1:2:1) was added to the lyophilized Drug Product, the resulting solution was mixed with 1 mL Montanide using a T-connector and applying 50 fast mixing cycles. PSD values for MFI analyses are given in ECD (equivalent circle diameter) and a number-based distribution is given. It should be noted that MFI and laser diffraction are complementary techniques. Therefore, a direct comparison of average particle size values obtained by laser diffraction and by MFI cannot be performed.
TABLE-US-00008 TABLE 7 DP-6P Particle size D (0.5) values in μm. T = 0 T = 1 T = 2 T = 3 Prep 1 1.77 1.71 1.70 1.71 Prep 2 1.73 1.62 1.69 1.69 Prep 3 1.61 1.73 1.75 1.69 Average 1.70 1.69 1.71 1.70
TABLE-US-00009 TABLE 8 DP-7P Particle size D (0.5) values in μm. T = 0 T = 1 T = 2 T = 3 Prep 1 1.64 1.59 1.56 1.59 Prep 2 1.59 1.61 1.61 1.57 Prep 3 1.65 1.66 1.63 1.55 Average 1.63 1.62 1.60 1.57
[0187] Table 7 and 8 show that by using a reconstitution composition comprising 750 μL 0.1M citric acid+250 μL PG/EtOH/Cremophor EL 1:2:1 (i.e. 750 μL 0.1M Citric acid in water, 62.5 μL Propylene Glycol, 125 μL Ethanol and 62.5 μL Cremophor EL), for both DP-6P and DP-7P emulsions can be prepared that are stable for at least 3 hours.
Example 2
Introduction
[0188] Therapeutic efficacy of SLP vaccination in combination with CpG1826 has previously been demonstrated in mice carrying established TC-1 tumors, which express the oncogenic E6 and E7 proteins of HPV16 (Zwaveling et al., J. Immunol. (2002) 169:350-358). To assess whether SLPs retain functionality in the most optimal formulation identified in Example 1 (750 μL 0.1M citric acid+250 μL PG/EtOH/Cremophor EL 1:2:1), we therapeutically vaccinated mice carrying a TC-1 tumor with an SLP harboring the D&-restricted CTL epitope RAHYNIVTF (represented herein by SEQ ID NO: 67), reconstituted either in DMSO/WFI or the novel reconstitution composition. All vaccines were subsequently emulsified in Montanide. Tumor outgrowth was monitored for 75 days. At the peak of the vaccine-induced T cell response, the percentage and phenotype of RAHYNIVTF-specific CD8.sup.+ T cells was determined in the blood. SLP reconstituted in DMSO/WFI and the novel reconstitution composition showed a similar potency in inducing TC-1 tumor regression. Mice vaccinated with the SLP reconstituted in the novel solution showed a higher percentage of RAHYNIVTF-specific CD8.sup.+ T cells in the blood.
Materials
[0189]
TABLE-US-00010 TABLE 9 Materials applied during TC-1 tumor experiment. Material Origin/supplier C57BL/6 female mice, Harlan Laboratories 6-8 weeks old Montanide ISA VG51 Seppic; batch 2384535/ U40740; exp 13 Feb. 2017 CpG ODN1826 (5 mg/ml) Invivogen; cat no tlrl-1826 DMSO Mylan; lot nr 140706; exp June 2017 WFI Fresenius Kabi; W005 4B03; exp 6 Mar. 2018 KLRG1-PeCy7 eBioscience; cat nr 25-5893-82 CD62L-Alexa780 eBioscience; cat nr 47-0621-82 CD44-Pacific Blue BioLegend; cat nr 103020 CD127-Biotin eBioscience; cat nr 13-1271-85 CD8a-Alexa700 eBioscience; cat nr 56-0081-82 CD3-V500 BD; cat nr 560771 Streptavidin- ThermoFischer; cat nr Qdot605 Q10101MP 7-AAD viability ThermoFisher, cat nr staining A1310; exp 22 Sep. 2016 D.sup.b-RAHYNIVTF Production of LUMC tetramer Trypsin Gibco (Life Technologies) cat nr 25200-056 Geneticin (G418) Gibco (Life Technologies) cat nr 10131-027 BSA Roche Diagnostics; cat nr 10735078001 Lysis buffer LUMC Pharmacy T-connector B Braun; 16494C Discofix C NORM-JECT Luer- HSW; 4010-000V0 lock 2 ml syringes NORM-JECT Luer- HSW; 4010-200V0 lock 1 ml syringes BD Microlance 3; BD; cat nr 300600 25G (0.5 × 16 mm) Disposables Various; LUMC
Methods
Vaccine Preparation
[0190] The following groups of mice were included in the study: [0191] Group 1: (n=5) 40% v/v DMSO/WFI emulsified 1:1 with Montanide ISA VG51. [0192] Group 2: (n=5) Reconstitution composition (750 μL 0.1M Citric acid in water, 62.5 μL Propylene Glycol, 125 μL Ethanol and 62.5 μL Cremophor EL per mL) emulsified 1:1 with Montanide ISA VG51. [0193] Group 3: (n=10) SLP GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR and 20 μg CpG ODN1826/mouse dissolved in 40% v/v DMSO/WFI, emulsified 1:1 with Montanide ISA VG51. [0194] Group 4: (n=10) SLP GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR (SEQ ID NO: 6) and 20 μg CpG ODN1826/mouse dissolved in Reconstitution composition (750 μL 0.1M Citric acid in water, 62.5 μL Propylene Glycol, 125 μL Ethanol and 62.5 μL Cremophor EL per mL), emulsified 1:1 with Montanide ISA VG51.
[0195] For mice in Group 1, a solution was prepared by admixing and subsequently swirling 400 μL DMSO and 600 μL WFI. The solution was taken up in a 2 mL Luer-Lock syringe (Syringe A). In another 2 mL Luer-Lock syringe (Syringe B) 1 mL of Montanide ISA VG51 was taken up, after which both syringes were connected to a T-connector. An emulsion was generated by mixing the contents back and forth extensively. After mixing, the syringes were disconnected and a 25G needle was placed on the syringe containing the emulsion. Per mouse, 100 μL was injected in the left flank subcutaneously.
[0196] The vaccine prepared for Group 2 was prepared in an identical manner, only differing by the use of reconstitution composition (750 μL 0.1M citric acid in water and 250 μL PG/EtOH/Cremophor EL 1:2:1, i.e. 0.075M citric acid, 6.25% v/v propylene glycol CAS no. 57-55-6, 12.5% v/v ethanol and 6.25% v/v polyoxyethyleneglyceroltriricinoleate 35 CAS no. 61791-12-6 in water) instead of DMSO and WFI. The vaccine for Group 3 was prepared by first dissolving the contents of a vial containing 1.5 mg SLP represented herein by SEQ ID NO: 6 (GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR) in 400 μL DMSO. The SLP was produced via Fmoc solid phase peptide synthesis (Fmoc Solid Phase Peptide Synthesis, A Practical Approach, W. C. Chan, P. D. White Eds, Oxford Univ. Press 2000). Then, 520 μL WFI and 80 μL CpG ODN1826 (stock 5 mg/ml) were added to the peptide in DMSO. After taking up this solution in a 2 mL Luer-Lock syringe, the same vaccine preparation protocol was followed as for Group 1 by emulsifying with Montanide ISA VG51. The preparation of vaccine for Group 4 was identical to the protocol for Group 3, only differing in the first step in which the contents of a vial containing 1.5 mg SLP SEQ ID NO: 6 were dissolved in 920 μL Reconstitution composition and adding 80 μL CpG ODN1826 (stock 5 mg/ml).
Therapeutic Vaccination
[0197] TC-1 tumor cells, expressing the oncogenic E6 and E7 proteins of HPV16 were cultured in complete IMDM culture medium, supplemented with 400 μg/ml geneticin. On day 0, TC-1 cells were harvested using trypsin and washed 3 times with PBS/0.1% BSA. Directly after harvesting, 100,000 TC-1 cells were injected s.c. in the right flank of 40 female C57BL/6 mice. On day 8, all mice were s.c. vaccinated in the left flank as described in the section Vaccine preparation. The tumor size of all mice was monitored at least twice a week using a caliper up to 75 days after tumor challenge. The study was carried out as displayed in
Measurement of Strength of T Cell Response in Blood
[0198] On day 9 after vaccination, blood was drawn from the tail vein of all mice. Blood samples were transferred to a 96-wells culture plate and centrifuged for 5 minutes at 1600 rpm. Erythrocytes were lysed by suspending blood cell pellets in Lysis buffer until orange coloration was observed. Subsequently, cells were washed in FACS buffer and stained with the fluorescent antibodies, the D.sup.b-RAHYNIVTF-APC tetramer and 7-AAD mentioned in the Materials section above. After 30 minutes of incubation on ice, cells were washed and analyzed on a BD LSRII flow cytometer in the Leiden University Medical Center (Dept. of Rheumatology).
Results
Tumor Outgrowth Similar Between Vaccinated Groups
[0199] By monitoring the tumor size at least twice a week, a growth curve could be created for each individual mouse. In
Vaccine-Induced Tetramer-Positive CD8.SUP.+ T Cells
[0200]
[0201] Significant differences were determined using an unpaired t-test, resulting in a p-value of p=0.022 between group 3 and 4.
Expression of KLRG1 and CD62L Indicate Favourable Antitumor Expression Profile after Vaccination with SLP 6
[0202] A study by Van Duikeren et al. (J Immunol, 2012; 189(7): 3397-403) aimed to identify parameters that correlated with the induction of an effective antitumor response. By identifying such biomarkers, different vaccine compositions can be tested in non-tumor bearing mice with prognostic value in tumor models. The authors found a correlation between the expression of KLRG1 and absence of CD62L expression on the one hand and effective antitumor immune responses on the other hand. We determined the percentages of KLRG1- and CD62L-expressing D.sup.b-RAHYNIVTF.sup.+ CD8.sup.+ T cells in the blood of vaccinated mice on day 9 after vaccination using flow cytometry. No difference in percentage of RAHYNIVTF-specific KLRG1.sup.+ CD62L.sup.− CD8.sup.+ T cells is observed between groups 3 and 4. Not enough RAHYNIVTF-specific CD8+ T cells were detected to reliably study the expression of KLRG1 and CD62L in the groups of mice vaccinated with vehicle only (Group 1 and 2). See Table 10 for the average percentages and standard deviations per group.
TABLE-US-00011 TABLE 10 Average percentages and SD of tetramer.sup.+ CD8.sup.+ T cells, and averages and percentages of expression of CD62L and KLRG1 of tetramer.sup.+ CD8.sup.+ T cells in groups of mice vaccinated with SLP. % of TM+ CD8 T cells % Tm+ % CD62L− % CD62L+ Group of CD8 KLRG1+ KLRG1+ 1 Average 0.1 # # SD 0 2 Average 0.1 # # SD 0.1 3 Average 1.4 51.9 4.0 SD 1.0 14.9 3.2 4 Average 4.8* 52.2 4.0 SD 3.8 16.6 2.0 *indicates significant difference (p < 0.05) between groups 3 and 4 as determined by unpaired t-test.
Discussion
[0203] No differences were observed in overall tumor outgrowth between the groups of mice vaccinated with SLP 6 dissolved either in DMSO/WFI or Reconstitution composition. We did observe enhanced induction of specific CD8+ T cells in the mice vaccinated with the SLP dissolved in Reconstitution composition as compared to the group of mice vaccinated with the SLP dissolved in DMSO/WFI.
[0204] The adjuvanting properties of Montanide have been ascribed to the formation of an antigen depot and induction of local inflammation and cell death, which favors maturation of antigen-presenting cells. The enhanced induction of tetramer.sup.+ CD8.sup.+ T cells in the group of mice vaccinated with the SLP dissolved in Reconstitution composition suggests that the combination of this solution with Montanide constitutes an emulsion with beneficial antigen release properties or local stimulation of antigen-presenting cells. The favourable profile of KLRG1 expression and absence of CD62L was similar between both groups of mice vaccination with the SLP. The data demonstrate that SLPs reconstituted in the reconstitution composition of the invention maintain their immunogenic capacity as compared to the originally used reconstitution composition (DMSO/WFI).
Example 3
Material
[0205] The following lyophilized peptide composition was used:
[0206] P53 DP5P: comprising peptides represented herein by SEQ ID NO: 191, 193, 194, 201 and 203.
[0207] The following chemicals were used: Cremophor EL. (Sigma Aldrich. Kolliphor EL); Propylene Glycol (≥99.5%. Sigma Aldrich); Ethanol (Absolute. VWR Emprove® Ph Eur. BP.USP); Citric acid (≥99%. Sigma Aldrich); MilliQ water; Sterile Montanide ISA 51VG (SEPPIC.)
[0208] The following equipment was used: Syringe extrusion devices (Discofix-3 T-connector. B. Braun); DMSO-resistant syringes (2 mL NORM-JECT Luer Lock. Henke Sass Wolf); Waters UPLC/MS system EQP-004; Protein Simple MFI 5200
Methods
[0209] Preparation of the vaccine emulsion and a placebo emulsion was performed as described in Table 1.
[0210] Analysis of chemical stability was performed by UPLC-MS as described in Example 1, at the section describing methods for analysis of in-use chemical stability of HPV-DP-6P and HPV-DP-7P vaccine emulsions including extraction of the peptides from the vaccine emulsion.
[0211] Particle size analysis was performed by Micro Flow Imaging. Prior to analysis a dilution of the vaccine emulsion was prepared by adding 10 μL of emulsion to 10 mL Reconstitution Solution and mixing until homogeneous, followed by 1:500 dilution of this solution in Reconstitution Solution.
[0212] Analysis settings of MFI 5200:
[0213] Method: DS500.2
[0214] Sample volume: 1 mL
[0215] Purge volume: 0.20 mL
[0216] Analysis: 0.68 min or 1.000.000 particles
[0217] Consecutive runs: 1
[0218] Results are expressed in Equivalent Circle Diameter (ECD) and a number-based distribution is given. Particles ≥15 μm are filtered from the results since these are known to be artefacts rather than emulsion particles.
Results
Purity of Reconstituted Drug Product
[0219] Purity of the Drug Product at different time points was calculated as follows:
Purity (%)=100%−Sum of impurities≥0.05% area
[0220] An overview of the in-use purity of the P53-DP-5P vaccine product is given in Error! Reference source not found.
TABLE-US-00012 TABLE 11 Overview of purity of reconstituted P53- DP-5P during storage at room temperature. PRODUCT: P53-DP-5P IN-USE STORAGE TIME TEST t = 0 h t = 1 h t = 2 h t = 3 h Purity 93.4 91.6 90.9 89.8 [Area %] Total related 6.6 8.4 9.1 10.2 substances (≥0.05%) [Area %]
[0221] As can be seen from Error! Reference source not found., purity of the Drug Products slowly decreases but is still ≥90.0% two hours after vaccine preparation. Example chromatograms of UPLC analysis of the vaccine at t=0 and t-=2 h are presented in
[0222] Identification of main peaks and impurities with an area ≥1.0% area was performed using mass spectrometry. All related substances with an area ≥1.0% area are reported and identified by comparing the measure m/z values with molecular masses of the peptide sequences and their known and expected modifications. The resulting overview of related substances for an in-use storage of P53-DP-5P for up to 3 hours is given in Table 12.
TABLE-US-00013 TABLE 12 Overview and identification of related substances of reconstituted P53-DP-5P. In-use stability up to 3 h after reconstitution. IN-USE STORAGE TIME SEQ RETENTION t = t = t = t = ID TIME (min.) 0 h 1 h 2 h 3 h NO Peptides and 4.97 13.00 12.02 11.87 11.63 194 related 7.99 12.43 11.73 11.72 11.60 201 substances 9.57 23.90 22.38 22.31 22.19 193 (≥0.05%) 14.14 30.37 28.75 28.81 28.81 191 [Area %] 16.82 < 1.05 1.54 1.87 203 intramo- lecular disulfide 19.46 13.73 16.73 16.21 15.56 203
Recovery of Drug Product from the Emulsion
[0223] The recovery of the five individual peptides present in P53-DP-5P from the emulsion was verified by comparison of emulsified and non-emulsified sample signals. An overview of the results is given in Error! Reference source not found.
TABLE-US-00014 TABLE 13 Overview of recovery by comparison of emulsified and non-emulsified sample signals. IN-USE STORAGE TIME SEQ t = 0 h t = 1 h t = 2 h t = 3 h ID NO Recovery 97 (0.6) 97 (0.8) 96 (2.4) 94 (2.3) 194 (RSD) 101 (2.0) 103 (3.8) 104 (5.1) 102 (5.1) 201 Both 98 (0.6) 99 (1.1) 99 (1.9) 98 (1.3) 193 values 96 (0.3) 98 (1.4) 99 (1.7) 98 (1.1) 191 given as % 70 (8.6) 92 (3.4) 90 (4.4) 86 (4.8) 203
Physical Stability
[0224] Physical stability was analysed by particle size analysis with MFI. Results are expressed in Equivalent Circle Diameter (ECD). Mean particle size values are given in Table 14, calculated from a number-based distribution.
TABLE-US-00015 TABLE 14 Mean particle size (ECD in μm) of P53 DP5P vaccine emulsions T = 0 h T = 1 h T = 2 h T = 3 h Prep 1 1.91 1.95 1.93 2.01 Prep 2 1.85 1.90 1.92 1.93 Average 1.88 1.93 1.93 1.97
Conclusion
[0225] Dissolution was successfully performed for a mixture containing 5 SLPs derived from the P53 antigen (P53 DP-5P).
[0226] Both chemical and physical in-use stability of the vaccine product was studied. Analysis of related substances and calculation of purity as summarized in Table 11 for P53 DP-5P shows that the purity of the Drug Product is ≥90.0% two hours after vaccine preparation. Only one related substance with a peak area % of ≥1% was observed. MS-identification showed that this peak is the intramolecular disulfide of the peptide set forth in SEQ ID NO: 203.
[0227] Physical stability of the P53 DP-5P vaccine product was studied by monitoring its particle size with MFI. The results of the particle size analysis are summarized in Table 14 and show that the particle size does not change up to three hours after vaccine preparation. In addition, all vaccine products were monitored by visual inspection during the stability study and no phase separation was observed at any time point.
Example 4
Material and Methods
[0228] The following lyophilized peptide composition was used:
PRAME DP5P: comprising peptides represented herein by SEQ ID NO: 153, 155, 156, 160 and 166:
[0229] A set of five PRAME-derived peptides was selected based on UPLC retention times, variation in amino acid composition, and solubility in reconstitution solution as determined by visual inspection.
[0230] Other materials and methods used were the same as in Example 3.
Results
Purity of Reconstituted Drug Product
[0231] Purity of the Drug Product at different time points was calculated as follows:
Purity (%)=100%−Sum of impurities≥0.05% area
[0232] An overview of the in-use purity of the PRAME-DP-5P vaccine product is given in Table 15. It should be noted that the purity of lyophilized PRAME-DP-5P is already below 90%. Nevertheless, the very limited decrease in purity over time demonstrates good chemical stability of this reconstituted drug product.
TABLE-US-00016 TABLE 15 Overview of purity of reconstituted PRAME- DP-5P during storage at room temperature. PRODUCT: PRAME-DP-5P IN-USE STORAGE TIME TEST t = 0 h t = 1 h t = 2 h t = 3 h Purity 82.9 83.7 82.5 82.2 [Area %] Total related 17.1 16.3 17.5 17.8 substances (≥0.05%) [Area %]
[0233] The low purity decrease over time indicates high chemical stability. The impurities with an area ≥1.0% area in PRAME-DP-5P were already present in the mixture before reconstitution. Since no significant increase of these impurities was observed in this stability study, no identification of the impurities was performed. The resulting overview of related substances for an in-use storage of PRAME-DP-5P for up to 3 hours is given in Table 16.
TABLE-US-00017 TABLE 16 Overview and identification of related substances of reconstituted PRAME-DP-5P. In-use stability up to 3 h after reconstitution. IN-USE STORAGE TIME SEQ RETENTION t = t = t = t = ID TIME (min.) 0 h 1 h 2 h 3 h NO Related 10.68 23.35 22.85 22.65 22.62 155 substances 17.50 13.61 13.49 13.34 13.40 160 (≥0.05%) 18.91 13.49 14.15 13.81 13.59 166 [Area %] 20.05 21.89 22.70 22.51 22.59 153 21.15 10.56 10.52 10.17 9.98 156
Recovery of Drug Product from the Emulsion
[0234] The recovery of the five individual peptides present in PRAME-DP-5P from the emulsion was verified by comparison of emulsified and non-emulsified sample signals.
[0235] An overview of the results is given in Table 17.
TABLE-US-00018 TABLE 17 Overview of recovery by comparison of emulsified and non-emulsified sample signals. IN-USE STORAGE TIME SEQ t = 1 h t = 1 h t = 2 h t = 3 h ID NO Recovery 81 (9.8) 87 (3.8) 84 (2.1) 81 (7.8) 155 (RSD) 81 (10.4) 87 (3.7) 84 (0.9) 82 (8.2) 160 Both 74 (10.8) 84 (3.9) 80 (1.0) 77 (8.1) 166 values 76 (10.7) 86 (3.3) 83 (0.2) 81 (8.6) 153 given as % 78 (10.5) 84 (3.6) 80 (1.0) 76 (7.8) 156
CONCLUSION
[0236] The purity of the PRAME DP-5P was not fully satisfactory (<90%), but the decrease in purity of the reconstituted vaccine product was very limited (purity T=0 82.9%, T=3 h 82.2%) confirming the benefits of the compositions described herein.