VIRUS AND ANTIGEN PURIFICATION AND CONJUGATION
20230056944 · 2023-02-23
Assignee
Inventors
- Leigh Burden (Owensboro, KY, US)
- Steven D. Hume (Owensboro, KY, US)
- Joshua Morton (Evansville, IN, US)
- Greg Pogue (Austin, TX, US)
- Barry Bratcher (Owensboro, KY, US)
- Hugh A. Haydon (Louisville, KY, US)
- Carrie A. Simpson (Evansville, IN, US)
- Nick Partain (Owensboro, KY, US)
- John W. Shepherd (Owensboro, KY, US)
Cpc classification
C12N2770/00051
CHEMISTRY; METALLURGY
C12N7/00
CHEMISTRY; METALLURGY
C07K2319/40
CHEMISTRY; METALLURGY
C12N2760/16134
CHEMISTRY; METALLURGY
C12N2770/40034
CHEMISTRY; METALLURGY
C12N2770/40051
CHEMISTRY; METALLURGY
C12N2760/16122
CHEMISTRY; METALLURGY
C12N2770/00041
CHEMISTRY; METALLURGY
International classification
Abstract
Disclosed herein are methods of forming compounds and exemplary compounds in the nature of a conjugated compound demonstrating enhanced stability, which in some embodiments comprises a protein and virus particle mixed in a conjugation reaction to form a conjugate mixture, such that the conditions and steps of forming these products allow for unrefrigerated storage for longer time periods than previous approaches, thus making feasible access to such products over a global supply chain.
Claims
1. A chemical compound, comprising: a protein conjugated to a virus particle, and wherein when the chemical compound is placed in an unrefrigerated environment at a storage temperature for a time period, an integrity or a concentration of the chemical compound at the end of the time period is at least 90% of an initial integrity or an initial concentration of the chemical compound, wherein the time period is at least 42 days a release date of the chemical compound.
2. The chemical compound of claim 1, wherein the protein is chemically associated with lysine residues on a surface of the virus particle.
3. The chemical compound of claim 1, wherein the storage temperature is at least 20° C.
4. The chemical compound of claim 1, wherein the virus particle is a virus.
5. The chemical compound of claim 4, wherein the virus is an enveloped virus.
6. The chemical compound of claim 4, wherein the virus is tobacco mosaic virus.
7. The chemical compound of claim 1, wherein the time period is at least 90 days after the release date of the chemical compound.
8. The chemical compound of claim 1, wherein the time period is at least 180 days after the release date of the chemical compound.
9. A chemical compound, comprising: a conjugated protein and a virus wherein the protein is chemically associated with lysine residues on a surface of the virus, and wherein when the chemical compound is placed in an unrefrigerated environment at a storage temperature for a time period, an integrity or a concentration of the chemical compound at the end of the time period is at least 90% of an initial integrity or an initial concentration of the chemical compound, wherein the time period is at least 42 days a release date of the chemical compound.
10. The chemical compound of claim 9, wherein the storage temperature is at least 20° C.
11. The chemical compound of claim 9, wherein the virus is tobacco mosaic virus.
12. The chemical compound of claim 9, wherein the protein is an antigen.
13. The chemical compound of claim 12, wherein the antigen is hemagglutinin antigen.
14. The chemical compound of claim 9, wherein the time period is at least 180 days after the release date of the chemical compound.
15. A chemical compound, comprising: an antigen and a virus particle wherein the antigen is chemically associated with lysine residues on a surface of the virus particle, and wherein when placed in an unrefrigerated environment at a storage temperature and after a time period of at least 42 days following a release date of the chemical compound, the chemical compound demonstrates a stability that exceeds an initial stability of the chemical compound stability for the antigen alone as measured by one or more of antigen concentration, antigen integrity, or antigen potency.
16. The chemical compound of claim 15, wherein the storage temperature is at least 20° C.
17. The chemical compound of claim 15, wherein the antigen is hemagglutinin antigen.
18. The chemical compound of claim 15, where the virus particle is tobacco mosaic virus.
19. The chemical compound of claim 15, wherein the time period is at least 180 days after the release date of the conjugate mixture.
20. The chemical compound of claim 15, wherein the measure of stability is antigen concentration, and a difference between the concentration of the conjugate mixture and the concentration of the antigen alone is at least 10%.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0033] The drawings and embodiments described herein are illustrative of multiple alternative structures, aspects, and features of the multiple embodiments and alternatives disclosed herein, and they are not to be understood as limiting the scope of any of these embodiments and alternatives. It will be further understood that the drawing figures described and provided herein are not to scale, and that the embodiments are not limited to the precise arrangements, depictions, and instrumentalities shown.
[0034]
[0035]
[0036]
[0037]
[0038]
[0039]
[0040]
[0041]
[0042]
[0043]
[0044]
[0045]
[0046]
[0047]
[0048]
[0049]
[0050]
[0051]
[0052]
[0053]
[0054]
[0055]
[0056]
[0057]
[0058]
[0059]
[0060]
[0061]
[0062]
[0063]
[0064]
[0065]
[0066]
[0067]
[0068]
[0069]
[0070]
[0071]
[0072]
[0073]
MULTIPLE EMBODIMENTS AND ALTERNATIVES
[0074] A multi-set process according to multiple embodiments and alternatives herein improves upstream purification processes, further enriching plant viruses, and facilitates the conjugation of virus and antigen to form a vaccine. Steps for producing and purifying a virus in accordance with multiple embodiments and alternatives are listed and discussed in connection with Table 1 and
Virus Production and Purification
[0075] Table 1 and
TABLE-US-00001 TABLE 1 Production and Purification of Virus Operative Unit In-Process Steps Operations In-Process Controls Analytics 1 Plant Growth Irrigation, Light Plant Height, (25 DPS) Nb Cycle, Fertilizer, structure and Media, Humidity, leaf quality Temperature 2 Infection Inoculum N/A with virus Concentration, Rate of Application 3 Viral Irrigation, Light N/A Replication Cycle, Humidity, (7 DPI) Plant Temperature Growth 4 Harvest of Visual Inspection of N/A Aerial Tissue Plants 5 Disintegration Blade Type and RPM, pH, Conductivity, of Plant Cells Screen Sizes, SDSPage, (Extraction) Buffer:Tissue Ratio Endotoxin, Nicotine 6 Clarification Ceramic Size, TMP, pH, Conductivity, of Plant kg/m.sup.2 SDSPage, Extract Endotoxin, Nicotine 7 Concentration Pore Size, TMP, Pore pH, Conductivity, of Clarified Material, kg/m.sup.2 SDSPage, Plant Extract Endotoxin, Nicotine 8 Ion-Exchange kg/L, Bed Height, pH, Conductivity, Chroma- Residence Time SDSPage, tography Endotoxin, Nicotine 9 Multi-Modal kg/L, Bed Height, pH, Conductivity, Chroma- Residence Time SDSPage, tography Endotoxin, Nicotine 10 Concentration Pore Size, TMP, Pore UV260, TEM, of Purified Material, kg/m.sup.2 DLS, SDSPage, Virus Endotoxin, Nicotine, Amino Acid
[0076] This purification platform is designed for commercial scalability and compliance with the cGMP regulations and utilizes one buffer throughout the entire purification process. According to multiple embodiments and alternatives, the steps of the virus purification platform are given in connection with plant expression. However, steps after the aerial tissue harvesting and cell rupture as described below also would apply to non-plant viruses (except where context is clearly related to plants, e.g., reference to removal of plant fiber).
[0077] In accordance with multiple embodiments and alternatives described herein, virus expression is accomplished through methods that are appropriate for a particular host. In some embodiments, virus-based delivery of genes to a plant host is accomplished with a modified TMV expression vector that causes tobacco plants to recombinantly form the virus. One such available alternative is the GENEWARE® platform described in U.S. Pat. No. 7,939,318, “Flexible vaccine assembly and vaccine delivery platform.” This transient plant-based expression platform described in this patent employs the plant virus TMV to harness plant protein production machinery, which expresses a variety of viruses in a short amount of harvest time post inoculation (e.g., less than 21 days). Tobacco plants inoculated with the virus genes express the particular virus in infected cells, and the viruses are extracted at harvest. Inoculation occurs by, as examples to be selected by a user of the methods herein described, hand inoculation of a surface of a leaf, mechanical inoculation of a plant bed, a high pressure spray of a leaf, or vacuum infiltration.
[0078] Besides Nicotiana benthamiana, other plant and non-plant hosts are contemplated by this disclosure, including those mentioned in the Summary. Besides the GENEWARE® platform, other strategies can be employed to deliver genes to plant (Lemna gibba or Lemna minor as non-limiting examples) and non-plant organisms (algae as a non-limiting example). These other strategies include Agro-infiltration, which introduces the viral gene via an Agrobacterium bacterial vector to many cells throughout the transfected plant. Another is electroporation to open pores in the cell membranes of the host to introduce the genes that recombinantly produce the viruses and antigens such as but not limited to those described in Examples 1 and 3 below. Another is TMV RNA-based overexpression (TRBO) vector, which utilizes a 35S promotor-driven TMV replicon that lacks the TMV coat protein gene sequence, as described in John Lindbo, “TRBO: A High-Efficiency Tobacco Mosaic Virus RNA-Based Overexpression Vector,” Plant Physiol. Vol. 145, 2007.
[0079] In some embodiments, growth of Nicotiana benthamiana wild type plants occurs in a controlled growth room. Plant growth is controlled via irrigation, light, and fertilized cycles. Plants are grown in a soilless media and temperature is controlled throughout the process.
[0080] After an appropriate number of days post sow (DPS), for example 23-25 DPS, the plants are infected with the virus replication. After infection, the plants are irrigated with water only and controlled via light cycle and temperature for a certain number of days post infection (DPI) depending on the type of virus.
[0081] Plants are inspected for height, infection symptoms, and the aerial tissue is harvested.
[0082] Virus recovery/cell rupture involves a disintegrator configured with an optimized blade/screen size followed by removal of residual cellulosic plant fiber from aqueous liquid (such as through a screw press, as one example).
[0083] An appropriate extraction buffer (e.g., 200 mM Sodium Acetate, pH 5.0; step 201 of
[0084] Ceramic permeate is further clarified via the use of glass fiber depth filtration (step 203 of
[0085] Clarified extract is concentrated with a TFF system (available from Sartorius AG). Cassette pore size (100-300 kDa), an appropriate TMP as described herein, and load of clarified extract per square meter of membrane surface area are controlled.
[0086] The clarified extract is concentrated to NMT 2× the ion-exchange column volume and washed 7× with ion-exchange chromatography equilibration buffer (200 mM Sodium Acetate, pH 5.0, step 204 of
[0087] The flow through and wash fractions are collected, combined and prepared for multi-modal Capto® Core 700 chromatography. The multi-modal chromatography column is equilibrated with five column volumes of equilibration buffer (200 mM Sodium Acetate, pH 5.0; step 206 of
[0088] The combined flow-through and wash fractions from Capto Q ion-exchange chromatography are loaded onto the column and the virus collected in the void volume of the column. The column is washed to baseline and stripped with high conductivity sodium hydroxide. Loading ratio, column bed height, residence time and chromatography buffers are all controlled. Formulation and concentration of virus (step 208,
[0089] All examples provided herein are meant as illustrative of various aspects of multiple embodiments and alternatives of any or all of virus production, virus purification, antigen production, antigen purification, and virus-antigen conjugation. These examples are non-limiting and merely characteristic of multiple alternative embodiments herein.
Example 1—Purification of Icosahedral Red Clover Mosaic Virus
[0090] The Western Blot, provided in
[0091]
[0092] Once the final step has occurred in the virus purification platform, the resulting viral product is highly purified, as shown by the visible band in lane 11 of
Example 2—Purification of Rod-Shaped TMV
[0093]
[0094] Accordingly, an inventive virus purification platform has successfully purified every virus on which the inventors have applied these methods, including both an icosahedral virus and a rod-shaped virus, and this platform is expected to be reproducible and consistently purify on a commercial scale virtually any type (if not all types) of virus.
Production and Purification of Recombinant Antigen
[0095] Table 2 and
TABLE-US-00002 TABLE 2 Production and Purification of Recombinant Antigen Operative In-Process Steps Unit Operations In-Process Controls Analytics 1 Plant Growth Irrigation, Light Cycle, Plant height, (25 DPS) Nb Fertilizer, Media, structure and Humidity, Temperature leaf quality 2 GENEWARE Inoculum Infection with Concentration, Rate Target Antigen of Application 3 Replication Irrigation, Light Cycle, (7-14 DPI) Humidity, Temperature Plant Growth 4 Harvest of Visual Inspection of Aerial Tissue Plants 5 Disintegration Blade Type and RPM, pH, of Plant Cells Screen Sizes, Conductivity, (Extraction) Buffer:Tissue Ratio SDSPage, Endotoxin, Nicotine 6 Clarification Filter Press Pore Size, pH, of Plant Extract Feed Pressure, kg/m2 Conductivity, SDSPage, Endotoxin, Nicotine 7 Concentration Pore Size, TMP, Pore pH, of Clarified Material, kg/m.sup.2 Conductivity, Plant Extract SDSPage, Endotoxin, Nicotine 8 Capto Q kg/L, Bed Height, pH, Chromatography Residence Time Conductivity, SDSPage, Endotoxin, Nicotine 9 ColMAC or kg/L, Bed Height, pH, ConA Residence Time Conductivity, SDSPage, Endotoxin, Nicotine 10 Ceramic kg/L, Bed Height, pH, Hydroxyapatite Residence Time Conductivity, SDSPage, Endotoxin, Nicotine 11 Concentration/ Pore Size, TMP, Pore UV260, TEM, Formulation Material, kg/m.sup.2 DLS, of Purified SDSPage, Antigen Endotoxin, Nicotine, Amino Acid
[0096] This purification platform is designed for commercial scalability and compliance with the cGMP regulations and utilizes one buffer throughout the entire purification process. According to multiple embodiments and alternatives, the steps of the antigen purification platform are as follows:
[0097] Growth of Nicotiana benthamiana wild type plants in a controlled growth room. Plant growth is controlled via irrigation, light and fertilizer cycles. Plants are grown in a soilless media and temperature is controlled throughout the process. After an appropriate number of DPS, for example 23 to 25, plants are infected for protein replication of a selected antigen. Once tagged, the protein is sufficient for retention in the ER of the transgenic plant cell. After infection plants are irrigated with water only and controlled via light cycle and temperature for an appropriate number of days post infection, such as 7-14 days depending on the type of antigen. Plants are inspected for height and infection symptoms, and the aerial tissue is harvested.
[0098] Recovery of antigen produced by the plants involves a disintegrator configured with an optimized blade/screen size followed by removal of residual cellulosic plant fiber from aqueous liquid (such as through a screw press, as one example).
[0099] A suitable extraction buffer is added to the resulting extract at an appropriate ratio, such as a 1:1 buffer:tissue ratio or a 2:1 buffer:tissue ratio. In some embodiments, the extraction buffer may be 50-100 mM Sodium Phosphate+2 mM EDTA+250 mM NaCl+0.1% Tween80, pH 8.5. Removal of chlorophyll and large cellular debris involves the use of filtration. Celpure300 is added at a ratio of 33 g/L and mixed for 15 minutes. Feed pressure (<30 PSI), filtrate pore size (0.3 microns), clarifying agent (Celpure300) and biomass loaded per square meter of membrane surface are all controlled to ensure passage of the antigens.
[0100] Clarified extract is concentrated with a TFF system (such as the Sartorius AG system). In some embodiments, the cassette pore size (for e.g., 30 kDa), an appropriate TMP as described herein, and load of clarified extract per square meter of membrane surface area are controlled.
[0101] The clarified extract is concentrated and washed 7× with an appropriate ion-exchange chromatography equilibration buffer (such as 50 mM Sodium Phosphate+75 mM NaCl, pH 6.5). The Capto Q ion-exchange column is equilibrated for five column volumes with 50 mM Sodium Phosphate+75 mM NaCl, pH 6.5, the feed is loaded, washed with equilibration buffer, and the column eluted/stripped with high salt.
[0102] Antigen fractions are collected in the elution for preparation for Cobalt IMAC chromatography. IMAC is equilibrated for five column volumes with 50 mM Sodium Phosphate+500 mM Sodium Chloride, pH 8.0, feed is loaded, washed with equilibration buffer and eluted using imidazole.
[0103] The elution fraction is diluted to conductivity, pH is checked and loaded onto a multi-modal ceramic hydroxyapatite (CHT) chromatography column. The CHT resin is equilibrated with five column volumes of equilibration buffer (5 mM Sodium Phosphate, pH 6.5). Antigens are eluted using a gradient of phosphate and NaCl. Loading ratio, column bed height, residence time and chromatography buffers are all controlled. Formulation and concentration of the antigens takes place using a TFF system (such as the Sartorius AG system). Pore size (in kDa), TMP, load per square meter of membrane surface area and pore material are all controlled, as further discussed herein.
[0104] Antigen is next concentrated to a suitable concentration, such as 3 mg/ml, and diafiltered with a suitable buffer (for example, phosphate buffered saline, pH 7.4). Formulated antigen is sterilized and stored appropriately. In some embodiments, sterilization is provided via a PES filter.
[0105]
Examples 3, 4, 5, and 6—H5 rHA, H7 rhA, WNV rDIII, and LFV rGP1/2
[0106] As shown in
Production of Recombinant Antigen—Virus Conjugates
[0107] Table 3 illustrates the steps of the conjugation of recombinant antigen according to multiple embodiments and alternatives.
TABLE-US-00003 TABLE 3 Production and Purification of Recombinant Antigen Operative In-Process In-Process Steps Unit Operations Controls Analytics 1 Concentration/ Pore Size, TMP, UV280 or BCA, Diafiltration of Pore Material, SDSPage, pH, Antigen kg/m.sup.2 Conductivity 2 Concentration/ Pore Size, TMP, UV260, SDSPage, Diafiltration of Pore Material, pH, Conductivity TMV 1295.10 kg/m.sup.2 3 Formulation of Mixing, Weight EDC Concentrate Check 4 Formulation of Mixing, Weight Sulfo-NHS Check Concentrate 5 Combine Antigen Molar Ratio, pH, Conductivity, and TMV 1295.10 Mixing, Volume SDSPage 6 Addition of EDC EDC Molarity, pH, Conductivity, Mixing, Volume SDSPage 7 Addition of Sulfo-NHS pH, Conductivity, Sulfo-NHS Molarity, Mixing SDSPage Volume 8 Conjugation Time, Temperature, Reaction Mixing 9 Reaction Time, Temperature, Quenching Mixing, Molarity of Amine Group 10 Diafiltration to Pore Size, TMP, pH, Conductivity, Remove Reactants Pore Material, SDSPage, kg/m.sup.2 Reactants (EDC/NHS) 11 Concentration/ Pore Size, TMP, Certificate of Formulation of Pore Material, Analysis Purified Vaccine kg/m.sup.2 (Drug Substance)
[0108] In an embodiment, the steps of a conjugation platform are as follows:
[0109] Purified antigen and virus are separately concentrated and diafiltered into a slightly acidic buffer, such as a 2-(N-morpholino) ethanesulfonic acid (MES) buffer containing NaCl.
[0110] A water soluble carbodiimide, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (known as EDC) is formulated in purified water to a molarity of 0.5 M.
[0111] A chemical reagent for converting carboxyl groups to amine reactive N-hydroxysulfosuccinimide esters, such as ThermoFisher's Sulfo-NHS, is formulated in purified water to a molarity of 0.1 M.
[0112] Antigen and virus are combined based upon weight or molarity and mixed to homogeneity (e.g. a 1:1 mg:mg addition).
[0113] The freshly prepared water soluble carbodiimide (such as EDC) is added to the mixture while mixing based upon molarity.
[0114] A chemical reagent for converting carboxyl groups to amine reactive esters (such as Sulfo-NHS) is added based upon molarity within one minute of EDC addition. The conjugation reaction begins and is continued until a predetermined mixing stop time, such as four hours, and the room temperature is controlled.
[0115] The reaction is quenched by adding free amines, and the chemical linker (for example EDC and Sulfo-NHS) is removed through a multi-modal chromatography step, such as Capto® Core 700, or diafiltration into a phosphate buffered saline. According to multiple embodiments and alternatives, the residual impurities are removed from the results of the conjugation reaction, sometimes referred to herein as a conjugate mixture, based on sized differences between impurities as the retentate, and the conjugate mixture as the permeate.
[0116] The conjugate mixture is diluted to target concentration. At this point, the virus-antigen conjugate is prepared for use as a purified vaccine/drug substance. A suitable delivery mechanism of the vaccine would include a liquid vial or lyophilized material to be reconstituted with physiologic buffering for project injection. Injection could be intramuscular or sub-cutaneous. Other delivery methods are contemplated, including without limitation intra-nasal.
Example 7—Conjugation of H7 rHA to TMV
[0117]
[0118] As shown in FIGS. 14-20, the conjugation platform of recombinant antigen to virus has successfully conjugated H7 rHA to TMV.
[0119] SEC-HPLC reports also indicated successful conjugation of H7 rHA to TMV in accordance with the current embodiments of the conjugation platform.
TABLE-US-00004 TABLE 4 SEC-HPLC Data of Free TMV Peak RT [min] Width [min] Area Height Area % Symmetry 13.233 0.77 1078.39 23.41 100 0.39
[0120]
TABLE-US-00005 TABLE 5 SEC-HPLC Data After H7 rHA is conjugated to TMV for 15 Minutes Peak RT [min] Width [min] Area Height Area % Symmetry 26.539 0.52 553.75 17.65 100 0.83
[0121]
TABLE-US-00006 TABLE 6 SEC-HPLC Data After H7 rHA is conjugated to TMV for 2 Hours Peak RT [min] Width [min] Area Height Area % Symmetry 13.304 0.73 37.30 0.86 0.36 0.43 20.569 1.83 167.16 1.52 1.59 0.00 22.336 1.17 62.55 0.89 0.59 0.64 24.489 2.05 73.35 0.60 0.70 1.34 26.510 0.54 10153.91 316.30 96.56 0.80 29.649 0.83 21.16 0.42 0.20 2.15
[0122] As illustrated in
[0123] As illustrated in
Example 8—UV Inactivation of TMV NtK
[0124] In order to avoid viral contamination of biopharmaceutical products, it is often necessary to inactivate (or sterilize) the virus to ensure the virus is no longer infectious. In addition, many regulatory agencies have enacted rules (such as the cGMP regulations) that require at least one effective inactivation step in the purification process of viral products. While UV-C radiation has been used in water treatment systems for many years, its use with biopharmaceutical products remains unexplored and there are limited studies regarding its ability to effectively inactivate viruses.
[0125] Accordingly, following virus production and purification but prior to conjugation with recombinant antigen, various UV-C conditions (i.e. energy density and wavelength) and various TMV concentrations were evaluated in order to effectively inactivate and sterilize TMV NtK. While many energy densities were tested, only the higher levels of energy densities successfully inactivated TMV NtK. In addition, it was determined that successful virus inactivation is concentration dependent because when the TMV solution was not diluted to an appropriate concentration, the UV-C irradiation did not effectively sterilize every virus in the sample. Therefore, the TMV solution must be appropriately dilute to permit the UV-C irradiation to interact with and effectively inactivate each virus.
[0126] As shown in
[0127] According to multiple embodiments and alternatives, the steps of the viral inactivation (following purification but before conjugation) are as follows:
[0128] Dilution of the TMV NtK solution to a concentration less than 50 micrograms/ml, as measured by A260 (which is a common method of quantifying nucleic acids by exposing a sample to UV light at a wavelength of 260 nm and measuring the amount of light that passes through the sample).
[0129] 0.45 micron filtration of the TMV solution to remove bacteria and any other large species that might interfere with UV line of sight.
[0130] Inactivating the TMV NtK by exposing the virus to light in the UV spectrum with an energy density between about 2400 J/m.sup.2 and about 5142 J/m.sup.2. In some embodiments, the energy density of the UV light is between about 4800 J/m.sup.2 and about 5142 J/m.sup.2. According to multiple embodiments and alternatives, the wavelength of the UV light is 254 nm.
[0131] Next, the inactivated TMV NtK is ready to be conjugated to the recombinant antigen.
[0132] These viral inactivation steps are designed for commercial scalability and compliance with the cGMP regulations
Example 9—pH Dependency of Conjugation
[0133] To evaluate whether incubating the virus at an acidic pH results in high quality conjugation, an experiment was performed using the same batches of virus, antigen, buffers, and esters, but changing only the formulation of the virus. In reaction 1, TMV was formulated into 1× MES Conjugation Buffer at pH 5.50 at a concentration of 3.1 mg/ml, according to multiple embodiments and alternatives. In reaction 2, TMV was concentrated to 11.0 mg/ml in phosphate buffer and added directly as 15% of the conjugation reaction volume. After these steps, the conjugation process was monitoring by SEC wherein an ordered decrease in free TMV from zero minutes (indicated by T=0) would indicate successful conjugation.
[0134] As shown in Tables 7 and 8, reaction 1 exhibited successful conjugation (due to the ordered decrease in free TMV from zero minutes) while reaction 2 was unsuccessful as shown by the percent remaining free TMV.
TABLE-US-00007 TABLE 7 Reaction 1, Successful Conjugation - TMV Formulated in Acidic pH Reaction 1 (TMV Formulated in Free TMV Peak Remaining Sample MES at 3.1 mg/mL) Area by SEC % Free TMV Free 284.8 nm 11104 N/A NtK T = 0 154.9 nm 9732 100% T = 5′ 139.8 nm 3909 40% T = 15′ 142.8 nm 1815 19% T = 30′ 149.4 nm 1039 11% T = 45′ 155.6 nm 769 8% T = 60′ 153.2 nm 777 8%
TABLE-US-00008 TABLE 8 Reaction 2, Unsuccessful Conjugation - TMV Formulated in Phosphate Buffer Reaction 2 (TMV at 11.0 mg/mL Free TMV Peak Remaining Sample in Phosphate Buffer) Area by SEC % Free TMV Free 64.2 nm 27590 N/A NtK T = 0 67.5 nm 14750 100% T = 5′ 68.8 nm 14916 101% T = 15′ 66.9 nm 13046 88% T = 30′ 73.3 nm 11705 79% T = 45′ 75.8 nm 8109 55% T = 60′ 80.0 nm 11020 75%
[0135] Accordingly, as shown in Table 7, incubation of the virus in acidic pH results in a conjugation greater than 90%. If the acidic pH incubation step does not occur, then the percent conjugation remains less than 50% (as shown in Table 8).
[0136] Based on this experiment, a model for conjugation (shown in
[0137] The virus activation steps, according to multiple embodiments and alternatives, are in contrast with traditional approaches in which the pH when storing viruses generally is maintained at or near neutral pH. As shown in
[0138] During the investigation of successful conjugations involving TMV, it was observed that successful conjugations generally occurred when the Dynamic Light Scattering (DLS)-measured radius of the virus increased during the activation step by at least a factor of 2.75 (see Table 9A, compared to Table 9B). In general, successful TMV conjugations (such as discussed with Table 9C) were characterized by an increase in DLS radius from about 70 nm to about 195 nm or higher, as shown in these tables.
[0139] Based on the successful conjugation which utilized virus activation, a platform was developed for conjugating purified antigen to purified virus. According to multiple embodiments and alternatives, the steps for preparing the purified antigen for conjugation are as follows:
[0140] To ensure pH control of the conjugation reaction, the purified antigen is formulated into a reaction buffer immediately prior to reaction initiation.
[0141] Prior to conjugation, purified antigens are stored in phosphate buffered saline at neutral to slightly basic pH.
[0142] The antigen pH target typically is pH 5.50 to 6.50, depending upon the nature of the molecule.
[0143] To facilitate conjugation to the virus, the storage buffer is replaced with a MES/NaCl buffer at acidic pH using ultrafiltration. The protein concentration is also increased to greater than 3 mg/mL.
[0144] The conjugation reaction is then initiated within four hours of antigen preparation completion to prevent destabilizing the protein structure.
[0145] According to multiple embodiments and alternatives, the steps for preparing the purified virus for conjugation are as follows:
[0146] After storage at neutral pH, the virus is activated at acidic pH prior to conjugation. For successful reactions, the virus is formulated from phosphate buffer at pH 7.4 into acetate buffer at pH 5.50 for a minimum of about 18 hours to a maximum of about 72 hours prior to the conjugation reaction start. In some embodiments, the virus is formulated from phosphate buffer at pH 7.4 into acetate buffer at pH 4.50 for a minimum of about 18 hours to a maximum of 72 hours prior to the conjugation reaction start. It was observed that storage of the virus for greater than 72 hours at acidic pH creates self-association between the viruses which causes virus insolubility and inhibits the efficiency of the conjugation.
[0147] Tables 9A and 9B further demonstrate the activation step in terms of increasing the radius of the virus (in this case, TMV) as measured by DLS. Specifically, Table 9A provides data for DLS radius increase of TMV after being activated, and before a successful conjugation occurred, with the antigens listed in the right-hand column. The “Factor by which radius increased” divides the TMV radius after activation by the typical TMV radius at neutral pH, which is about 70 nm. Conversely, Table 9B provides data for DLS radius increase of TMV after an activation step was started, in advance of unsuccessful attempts at conjugation, with the antigens listed in the right-hand column. In Tables 9A and 9B, the left column represents the standard radius of TMV rods at neutral pH and under general storage conditions, i.e., before any activation occurs.
TABLE-US-00009 TABLE 9A Free TMV radii as measured by DLS (Prior to successful conjugation) TMV radius after TMV radius at activation (nm) Factor by which neutral pH (DLS results) radius increased Antigen 70 nm 195.2 2.789 SG 70 nm 207.2 2.960 SG 70 nm 249.1 3.559 SG 70 nm 249.1 3.559 SG 70 nm 228.6 3.266 SG 70 nm 234.1 3.344 SG 70 nm 234.1 3.344 SG 70 nm 441.3 6.304 SG 70 nm 284.8 4.069 SG 70 nm 517.6 7.394 SG 70 nm 574.0 8.200 SG 70 nm 448.2 6.403 SG 70 nm 209.7 2.966 PH 70 nm 220.4 3.149 PH 70 nm 495.6 7.080 PH 70 nm 517.6 7.394 PH 70 nm 266.8 3.811 CO 70 nm 495.6 7.080 CO 70 nm 517.6 7.394 CO 70 nm 295.4 4.220 MI 70 nm 517.6 7.394 MI 70 nm 574.0 8.200 MI Average (nm): Average Factor 413.5 for Increase: 5.176
TABLE-US-00010 TABLE 9B Free TMV radii as measured by DLS (Prior to unsuccessful conjugation) TMV radius at TMV radius after neutral pH activation (nm) Factor by which (standard) (DLS results) radius increased Antigen 70 nm 95.4 1.363 SG 70 nm 105.4 1.506 SG 70 nm 156.0 2.229 SG 70 nm 176.5 2.521 PH Average (nm): Average Factor 133.3 for Increase: 1.905
[0148] Following these preparation steps, the antigen and virus reactants were mixed to form a conjugate mixture and the conjugation progress was monitored using DLS and SDS-PAGE methods. Table 9C illustrates the average molecular radius of the conjugation reaction over time using DLS after the virus was activated using acidic pH. As shown in Table 9C, molecular radius is one indicator of successful coating of the viral rods with antigen molecules.
TABLE-US-00011 TABLE 9C TMV NtK SEC and DLS History Soluble NTK SEC DLS Radius Peak Area (nm) 10750 496 9651 518 7106 574 5538 660
[0149] In turn,
Example 10—TEM Imaging of Different Ratios of Purified Virus to Purified Antigen for Conjugation
[0150] The desired conjugation reaction between purified virus and purified antigen is represented by the following formula:
Virus+Antigen.fwdarw.Virus-Antigen (Formula 1)
[0151] However, it is well known that antigens are prone to self-conjugation and the desired reaction may not be obtained, as shown by the following formula:
Virus+Antigen.fwdarw.Virus-Antigen+Antigen-Antigen (Formula 2)
[0152] Self-conjugation of the purified antigen is a problem for the successful development of vaccines because the antigen-antigen conjugates are not removed during the size chromatography step and the result is a minimized or reduced immune response.
[0153] To address this self-conjugation problem, various experiments were performed to determine how to consume the unreacted antigens and antigen conjugates. First, the antigens were capped by exposing them to reagents that inhibited self-conjugation. While it was anticipated that this traditional approach would be successful, this approach failed because the reaction occurred too quickly.
[0154] Next, the virus to antigen ratios were adjusted to determine suitable conjugation ratios. As shown in Tables 10 and 11 and
TABLE-US-00012 TABLE 10 TEM Imaging Samples - Control Groups Apprx. Volume Temp. Sample Description Lot (μl) Stored Concentration 1 HA Alone 19UL-SG- 100 4° C. 1.01 mg/ml 001 free HA 2 TMV NtK 18HA-NTK- 100 4° C. 0.54 mg/ml Alone 001 free TMV NtK 3 HA:HA 19UL-SG- 100 4° C. 2.335 mg/ml Conjugates 004 with added TMV NtK
TABLE-US-00013 TABLE 11 TEM Imaging Samples - Conjugates Approx. Volume Temp. Sample Ratio Lot (μl) Stored Concentration 4 TMV:HA = 18TAP-SG- 100 4° C. 5.2 mg/ml 1:1 002 5 TMV:HA = 19UL-SG-001 100 4° C. 1.688 mg/ml 1:1 6 TMV:HA = 19UL-SG-002 100 4° C. 1.387 mg/ml 4:1 7 TMV:HA = 19UL-SG-003 100 4° C. 3.479 mg/ml 16:1
[0155]
[0156]
[0157]
[0158]
[0159]
[0160]
[0161]
[0162]
Example 11—Sedimentation Velocity Analysis of Different Conjugation Conditions
[0163] Sedimentation velocity (“SV”), as measured in an analytical ultracentrifuge (“AUC”), is an ideal method for obtaining information about protein heterogeneity and the state of association of aggregation. Specifically, aggregates or different oligomers can be detected on the basis of different sedimentation coefficients. This method also detects aggregates or other minor components at a level below 1% by weight. Furthermore, SV provides high quality quantitation of the relative amounts of species and provides accurate sedimentation coefficients for any aggregates.
[0164] In order to measure the amount of self-conjugated and unreacted HA, as well as the amount of HA occupancy on TMV NtK with different conjugation conditions, the total signal associated with the sedimentation of free antigen, free virus, and various TMV:HA ratios were measured using SV-AUC. The following samples and descriptions are provided in Table 12:
TABLE-US-00014 TABLE 12 Samples and Descriptions for SV-AUC Sample Description Lot Concentration 1 HA Alone 19S-G-001 1.01 mg/ml 2 TMV Ntk Alone 18HA-NTK-001 0.54 mg/ml 3 TMV:HA = 1:1 19UL-SG-004 1.0 mg/ml 4 TMV:HA = 1:1 18TAP-SG-002 1.0 mg/ml 5 TMV:HA = 1:1 19UL-SG-001 0.8 mg/ml 6 TMV:HA = 4:1 19UL-SG-002 1.0 mg/ml 7 TMV:HA = 16:1 19UL-SG-003 1.0 mg/ml
[0165] These stocks were shipped cold (not frozen) and subsequently stored at 2-8° C. until analyzed. 1×PBS from Corning was used for sample dilution and as a reference blank. Sample 1 was diluted 1:1, and samples 2-7 were diluted 1:3 with 1×PBS to create the sedimentation velocity samples. These dilutions were carried out to bring the total absorbance of the sample within the linear range of the absorbance detection system.
[0166] Methods—The diluted samples were loaded into cells with 2-channel charcoal-epon centerpieces with 12 mm optical pathlength. 1×PBS was loaded into the reference channel of each cell. The loaded cells were placed into an analytical rotor, loaded into an analytical ultracentrifuge, and brought to 20° C. The rotor was then brought to 3000 rpm and the samples were scanned (at 280 nm) to confirm proper cell loading. For samples 2-7, the rotor was brought to the final run speed of 9,000 rpm. Scans were recorded at this rotor speed as fast as possible (every 3 min) for ˜11 hours (250 total scans for each sample). For sample 1 (the free HA), the rotor was brought to 35,000 rpm and scans were recorded every 4 min for 5.3 hours. The data was then analyzed using the c(s) method described in Schuck, P. (2000), “Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling,” Biophys. 1 78, 1606-1619. Using this method, raw scans were directly fitted to derive the distribution of sedimentation coefficients, while modeling the influence of diffusion on the data to enhance the resolution.
[0167] Results and Discussion—The high-resolution sedimentation coefficient distributions for samples 1-7 are shown in
[0168]
TABLE-US-00015 TABLE 13 Extent of trimerization Pre-Clinical HA lot SEC SEC Antigen Lot number Trimer % Monomer % B/Colorado 18TAP-CO- 18HA-CO- 55.05% 44.95% 001 003 A/Michigan 18TAP-MH- 18HA-MH- 11.93% 88.07% 002 007 B/Phuket 18TAP-PH- 18HA-PH- 84.51% 15.49% 002 003 A/Singapore 18TAP-SG- 18HA-SG- 94.52% 3.90% 002 003
[0169] As also shown in
[0170] In
[0171]
[0172]
[0173]
[0174]
[0175]
[0176] The results for the virus-antigen normalized sedimentation coefficient distribution, as shown in
TABLE-US-00016 TABLE 14 SV-AUC Results of the Different Virus-Antigen Conjugates Fraction Between Fraction Between 40-2000 S (%) 1-40 S (%) (HA (TMV NtK-HA Sample Lot Ratio monomer/trimer) Conjugate) 3 19UL-SG-004 1:1 37.1 62.9 4 18TAP-SG- 1:1 26.1 73.9 002 5 19UL-SG-001 1:1 31.4 68.6 6 19UL-SG-002 4:1 11.2 91.1 7 19UL-SG-003 16:1 0.6 99.4
[0177] The results in Table 14 indicate that a 1:1 ratio has more self-conjugation of HA and HA products, as compared to the 4:1 and 16:1 ratios. In addition, increasing the TMV:HA ratio results in virtually complete engagement of HA products in TMV-conjugation events (approaching almost 100% conjugation in sample 7).
[0178] According to multiple embodiments and alternatives, decreasing the amount of HA in a conjugation reaction, by increasing the TMV NtK to HA ratio from 1:1 to 16:1, results in: (1) reducing the aggregation of HA antigen on each TMV rod, as observed by Example 10 and
Example 12—Immune Response in Mice
[0179] To determine immune response following administration of the inventive virus-antigen conjugates, mice were administered the conjugates as vaccines via intramuscular injection. Each vaccine was a TMV:HA conjugate produced at a 1:1 (TMV:HA) ratio as described herein, administered to most of the animals on Day 0 and 14 of the study (control animals were administered buffer alone, TMV alone, or HA alone). Those administered vaccine received either 15, 7.5, or 3.75 mcg (micrograms) of antigen, as shown below in Table 15. One cohort had samples drawn on Day 7, another at Days 14 and 21, and a third at Days 28, 42, and 90, with the samples then subjected to hemagglutination inhibition (HAI) assay.
[0180] Based on the assay, no measurable response from any animal for any vaccine occurred at Days 7 or 14. However, initial responses were seen in some animals on Day 21. Specifically, 10/27 animals showed low level responses (only 1 of them >80 HAI titers) for H1N1 vaccine (Influenza A/Michigan/45/2015 (H1N1pdm09)). Also, 22/27 showed low level responses (only 2 of them >80) for H3N2 vaccine (Influenza A/Singapore/INFIMH-16-0019/2016). On Day 28, the number of animals within this cohort responding measurably to H1N1 vaccine was 8/29 with a single animal at 80 HAI titers and all others less. For H3N2 vaccine, the number responding measurably was 14/29, also with a single animal at 80 HAI titers and all others less.
[0181] The most pronounced results were observed from blood samples taken at Day 42 and Day 90, which are presented in Table 15, below. In this table, a standard error of the mean (SEM) is provided with the average and the fraction of animals responding (Fr.Resp.). It will be noted that in each cohort, some of the mice received vaccines for Influenza B viruses (B/Colorado/06/2017 (V) and B/Phuket/3073/2013 (Y), respectively). No response was detected in these animals on any of the days, as expected because B-type influenza viruses and corresponding HA immunogens are known to not generate HAI titers in mice with the efficiency and effectiveness as A-type HA immunogens.
TABLE-US-00017 TABLE 15 Immune response based on dose and time post-vaccination Day 42 Day 90 Average HAI Titers Average HAI Titers Immunogen H1N1 Fr. Resp H3N2 Fr. Resp H1N1 Fr. Resp H3N2 Fr. Resp 1. Vehicle 0 0 0 0 0 0 0 0 alone 2. TMV alone: 0 0 0 0 0 0 0 0 15 mcg 3. HA Quad 0 0 0 0 0 0 0 0 15 mcg 4. HA Quad 0 0 0 0 0 0 0 0 7.5 mcg 5. HA Quad 0 0 0 0 0 0 0 0 3.75 mcg 6. V-HA Quad 20 ± 5.477 7/10 27 ± 7.218 8/10 274 ± 66.336 10/10 136 ± 33.442 10/10 15 mcg 7. V-HA Quad 26 ± 4.733 9/10 22 ± 9.466 6/10 174 ± 40.797 9/10 84 ± 45.77 6/10 7.5 mcg 8. V-HA Quad 19 ± 4.566 7/9 17 ± 4.969 6/9 224 ± 62.993 8/9 40 ± 10.423 7/9 3.75 mcg
[0182] Separate from the previously described immune response study, and to further evaluate the inventive system in terms of suitable virus to antigen ratios, the humoral immune response in mice was evaluated following vaccination at various TMV:HA conjugate ratios (i.e., 1:1, 4:1, 16:1) of both Influenza A Antigen and Influenza B Antigen along with controls as noted below. In this manner, various conjugation ratios and their effect on immune response were studied. The mice receiving vaccination were administered 15 mcg HA via injection on Day 0 and Day 14 of the study, in a subcutaneous region dorsally The serum antibody responses to the vaccination were then analyzed for HA-specific activity. Tables 15 (H3 influenza virus used as capture protein) and 16 (recombinant H3 protein used as capture protein) show the groupings of mice (12 mice per grouping), and the agents that were administered, with the right-hand column in each table presenting ELISA antibody (Ab) titers results.
TABLE-US-00018 TABLE 16 TMV:HA ratio study - A-type influenza HA. Conjugation ratio Average ELISA Grouping Vaccine (TMV:Antigen) Ab Titer 1. Phosphate-buffered saline n/a 0 2. TMV-H3 H3 HA:HA 0 3. TMV-H3 1:1 0 4. TMV-H3 4:1 120 5. TMV-H3 16:1 200
[0183]
[0184] In addition to Influenza A H3 Antigen, Influenza B Antigen also was studied (B-Phuket HA) using the binding propensity of recombinant Influenza B Phuket Antigen and its corresponding antibody. Table 17, below, presents the results of this part of the study that was there is not as clear of a showing of 16:1>4:1>1:1 based on the results of average ELISA Ab titers.
TABLE-US-00019 TABLE 17 TMV:HA ratio study - B-type influenza HA. Conjugation ratio Average ELISA Grouping Vaccine (TMV:Antigen) Ab Titer 1. Phosphate-buffered saline n/a 0 2. TMV-B B Phuket HA:HA 283± 3. TMV-B 1:1 211± 4. TMV-B 4:1 56± 5. TMV-B 16:1 329±
[0185] Even so, the 16:1 ratio demonstrated the highest average antibody titer. Thus, the inventors believe it is reasonable to predict the same relationship between density and immune response applies to the study of the Influenza B Antigen (B-Phuket HA). That is, as with the results of H3 antigen, immune response will be higher for less dense forms of the conjugates. Additionally, there is reason to believe the conjugation reaction for the 4:1 ratio did not proceed as the reactions for the other ratios because of possible abnormalities during conjugation, and the fact that neither electron microscopy nor ultracentrifugation analysis were performed on this sample. In any case, the data here show immune response at all three ratios. The fact that immune response was achieved at multiple ratios underscores the robustness of the system for not being tied to any one particular ratio. This flexibility as seen with the particular TMV-conjugated vaccines probably gives further indication that the system will work well both when other antigens are conjugated to TMV besides the H3 and H1 antigens included in these studies, as well as when other virus carriers besides TMV are used for the carrier.
[0186] In terms of clinical utility, a product conjugated in accordance with any of multiple embodiments and alternatives described herein may be utilized as a vaccine by delivering the purified antigen via a purified virus, such as but not limited to the virus-antigen conjugates described in Examples 7, 9, 10, 11, and 12. Still further, embodiments of the present disclosure include any vaccine products packaged in any number of forms (e.g., vial) with appropriate buffers and additives, being manufactured from any virus-protein conjugate compositions, the conjugation of which is provided for herein. In this respect, embodiments include those wherein such vaccine products are amenable to delivery in the form of unit doses provided to a human or animal patient, such as but not limited to administration by syringe or spray through routes that include, but are not limited to, subcutaneous, intramuscular, intradermal administration, and nasal, as well as administration orally by mouth and/or topically, to the extent clinically indicated. By way of non-limiting example, and without detracting from the breadth and scope of the embodiments herein, the size of TMV (typically 18 nm×300 nm) and its rod-like shape promotes antigen uptake by antigen presenting cells (APCs), and thus serves to enhance immunity of T cells (such as Th1 and Th2) and provides adjuvant activity to surface conjugated subunit proteins. This activity is also stimulated through viral RNA/TLR7 interaction. As a result, the combined effect of vaccine uptake directly stimulates activation of the APCs. Humoral immunity is typically balanced between IgG1 and IgG2 subclasses through subcutaneous and intranasal delivery. Upon mucosal vaccine delivery, responses also include substantial systemic and mucosal IgA. Cellular immunity is also very robust, inducing antigen-specific secretion, similar to a live virus infection response. Whole antigen fusions allow for native cytotoxic T lymphocyte (CTL) epitope processing, without concern for human leukocyte antigen (HLA) variance.
[0187] The broad (humoral and cellular) and augmented (amplitude and effectiveness) immune responses associated with the multi-set purification platform according to current embodiments are in sharp contrast to subunit proteins tested without TMV conjugation, which induce little or no cellular or humoral immunity. The impact of these immune responses is that vaccines created via the multi-set platform, according to current embodiments, promotes highly protective responses as single dose vaccines and offers speed and safety not offered by other conventional vaccine platforms. Indeed, the conjugation platform is shown to work on a wide array of viruses and proteins (including antigens), combined within a broad range of ratios and successfully administered at various doses, which again are indicative of the robustness of the system. Additional advantages of the multi-set platform for producing vaccines in current embodiments include: a proactive antigen-stimulating approach for systemic immune protection against pathogen challenge, the platform is highly adaptable to produce antigenic domains from disease pathogens (including virus glycoproteins or non-secreted pathogen antigens), and the platform serves as an efficacious vaccine platform for both virus and bacterial pathogens.
[0188] In addition to advantages regarding vaccine applications, plant virus particles purified via the multi-set platform according to current embodiments can be formulated for various drug delivery purposes. These different purposes may include: 1) immune therapy—through the conjugation of therapeutic antibodies to the surface of virus particles and their delivery to enhance cytotoxic effect; 2) gene therapy—through loading specific nucleic acids for introduction into particular cell types for genetic modification, and 3) drug delivery—through loading chemotherapeutic agents into virus particles for targeted tumor delivery.
[0189] As a brief example of the many advantages of the methods discussed herein, the multi-set platform according to multiple embodiments could be utilized as a drug delivery tool by first causing the purified virus to swell by exposing it to a pH shift as discussed above. Subsequently, the virus in this condition would be incubated with a solution of concentrated chemotherapeutic agent, such as doxorubicin, and the pH is then reverted to neutral thereby causing the virus to return to its pre-swollen state and thereby entrapping the chemotherapeutic molecules. Next, the virus particle could be delivered to an organism by a delivery mechanism chosen from a group that includes, but is not necessarily limited to, injection for targeted treatment of tumors.
[0190] Accordingly, the above descriptions offer multiple embodiments and a number of alternative approaches for (i) the plant-based manufacture and purification of viruses; (ii) the plant-based manufacture and purification of antigens; and (iii) the formation of virus-antigen conjugates outside the plant that are therapeutically beneficial as vaccines and antigen carriers; and (iv) the delivery of therapeutic vaccines comprising a purified virus and purified antigen.
Example 13—Vaccine Stability Under Refrigerated and Room Temperature Conditions
[0191] Vaccines have dramatically improved human and animal health. For instance, in the 20.sup.th Century alone, vaccines have eradicated smallpox, eliminated polio in the Americas, and controlled a variety of diseases throughout the world. However, vaccines are highly unstable and very sensitive to changes in temperature. As discussed in F. Coenen et. al., Stability of influenza sub-unit vaccine. Does a couple of days outside the refrigerator matter? Vaccine 24 (2006), 525-531, influenza vaccines are generally unacceptable and inactive after five weeks at room temperature storage (i.e. ˜25° C.). Of all the influenza vaccines discussed in the F. Coenen article, only one vaccine exhibited stability for 12 weeks at room temperature storage. This is a significant problem with other vaccine types too. Accordingly, all current vaccines must generally be refrigerated during the entire supply chain from the moment of commercial production until administration, often referred to as the “cold chain.”
[0192] While in a refrigerated environment, the majority of vaccines remain stable for the typical seventy-eight week goal of stability. However, the absolute requirement for cold chain is a global problem that has limited the availability of vaccines worldwide because it is often difficult to guarantee in developing countries and has led to widespread vaccine loss. Many efforts have been made to create room temperature stable vaccines, but as discussed in the literature, those efforts have been unsuccessful. In addition, the cold chain is very costly to maintain for manufacturers, as well as the doctors and organizations receiving, storing, and applying the vaccines to populations. Accordingly, there is a significant and global need for increasing the stability of vaccines and enhancing vaccine-antigen stability in order to reduce the dependency on the cold chain and to ensure vaccines retain their potency until administration. In addition, improving stability can prolong the vaccine shelf life, which would facilitate the stockpiling of vaccines in the preparation of a potential pandemic and prevent vaccine loss in unfavorable conditions. Along with other features and advantages outlined herein, the scope of present embodiments meet these and other needs. In doing so, the inventive purification and conjugation platform extends the stability of protein-virus conjugates under both refrigerated and room temperature conditions.
[0193] There are several methods for determining antigen quality and vaccine stability including: (1) protein concentration as measured by BCA Protein assay (which is based on the principle that proteins can reduce Cu.sup.2+ to Cu.sup.+1 in an alkaline solution which results in a purple color formation), (2) storage potency as measured by VaxArray antibody array binding (which utilizes multiplexed sandwich immunoassays), (3) SDS-Page purity as measured in terms of a single migrating band, (4) pH as a measurement of the physical pollution properties, and when possible, (5) size exclusion chromatography to characterize the multimeric structure of the antigen. Moreover, a vaccine is considered unacceptable for use if it fails the BCA Protein assay, the VaxArray test, or the SD S-Page analysis. In other words, if a vaccine fails any one of these three tests, the vaccine is unacceptable for use and inactive.
[0194] Accordingly, the five tests mentioned in the previous paragraph were conducted on the following influenza HA antigens produced and purified in accordance with multiple embodiments and alternatives: H1N1 (A/Michigan), H3N2 (A/Singapore), H1N1 (A/Brisbane), H3N2 (A/Kansas), B/Colorado, and B/Phuket. The following tables provide the stability data and storage potency as measured at release and various times after filling into vials and stored under refrigerated conditions (4° to 8° C.). As used herein, an initial concentration or integrity refers to the concentration or integrity of a compound, conjugate mixture, pharmaceutical product, vaccine, or the like at its release date, and the release date is determined based on 21 C.F.R. Part 11 and ICH Q1A Stability Testing of New Drug Substances and Products, Revision 2 (November 2003), the full contents both of which are incorporated by reference herein.
TABLE-US-00020 TABLE 18 Stability of Purified H1NI (A/Michigan) Under Refrigerated Conditions Test Test Initial 1 3 4 5 6 Parameters Method Units (CoA) month months months months months Concentration BCA mg/mL 1.081 1.057 1.068 1.066 1.060 0.921 Purity SDS PAGE % 97% >99% 92% .sup. 88% .sup. 81% .sup. 76% Purity SEC Peak 1% 11.93% 6.18% 0.00% 2.83% 4.77% 4.92% Peak 2% 88.07% 93.82% 100.00% 97.17% 95.23% 95.71% Physical/Chemical pH NA 7.4 7.4 7.4 7.2 7.3 7.3 Properties Storage Potency VaxArray μg/mL 93 164 987 1300 1085 1176
TABLE-US-00021 TABLE 19 Stability of Purified H3N2 (A/Singapore) Under Refrigerated Conditions Test Test Initial 1 3 4 5 6 Parameters Method Units (CoA) month months months months months Concentration BCA mg/mL 0.855 0.900 0.891 0.908 0.885 0.795 Purity SDS PAGE % >99% >99% .sup. >99% .sup. >99% .sup. >99% .sup. >99% Purity SEC Peak 1% 94.52% 97.95% 100.00% 100.00% 100.00% 100.00% Peak 2% 3.90% 0.00% 0.00% 0.00% 0.00% 0.00% Physical/Chemical pH NA 7.4 7.4 7.4 7.2 7.3 7.3 Properties Storage Potency VaxArray μg/mL 746 671 1037 624 872 1089
TABLE-US-00022 TABLE 20 Stability of H1N1 (A/Brisbane) Under Refrigerated Conditions Test Test Parameters Method Units Initial (CoA) 1 month 3 months Concentration BCA mg/mL 0.804 0.810 0.967 Purity SDS % >99% 78% 73% PAGE Purity SEC Trimer % 20.85% Trimer 11.21% Trimer 100% single Monomer % 79.15% Monomer 88.79% Monomer peak Storage VaxArray μg/mL 1205 1064 768 Potency
TABLE-US-00023 TABLE 21 Stability of H3N2 (A/Kansas) Under Refrigerated Conditions Test Test Parameters Method Units Initial (CoA) 1 month 3 months Concentration BCA mg/mL 0.9 0.923 1.211 Purity SDS % 95% 93% 90% PAGE Purity SEC Trimer % 30.92% Trimer 5.20% Trimer 100% single Monomer % 69.08% Monomer 94.80% Monomer peak Storage VaxArray μg/mL 916 1061 1094 Potency
TABLE-US-00024 TABLE 22 Stability of B/Colorado Under Refrigerated Conditions Test Test Initial 1 3 4 5 6 Parameters Method Units (CoA) month months months months months Concentration BCA mg/mL 0.848 0.855 0.862 0.873 0.885 0.777 Purity SDS PAGE % 99% 63% 46% 40% 38% 35% Purity SEC Peak 1% 55.05% 39.70% 38.87% 20.77% 20.88% 39.55% Peak 2% 44.95% 49.86% 61.13% 79.23% 79.12% 60.45% Physical/Chemical pH NA 7.3 7.5 7.4 7.3 7.3 7.4 Properties Storage Potency VaxArray μg/mL 541 446 733 528 823 1082
TABLE-US-00025 TABLE 23 Stability of B/Phuket Under Refrigerated Conditions Test Test Initial 1 3 4 5 6 Parameters Method Units (CoA) month months months months months Concentration BCA mg/mL 0.957 0.895 0.912 0.951 0.818 0.819 Purity SDS PAGE % 96.1% >99% .sup. 97% 97% 93% .sup. 91% Purity SEC Peak 1% 84.51% 90.05% 91.98% 85.96% 85.76% 92.47% Peak 2% 15.49% 9.95% 8.02% 14.04% 14.24% 7.53% Physical/Chemical pH NA 7.4 7.4 7.3 7.3 7.3 7.4 Properties Storage Potency VaxArray μg/mL 910 945 888 952 812 924
[0195] Tables 18-23 illustrate that the purified free antigens exhibit different patterns of stability. For instance, some antigens like H1N1 (A/Michigan) and H3N2 (A/Singapore) appeared stable after 6 months with no significant deviations in measurements (as is typically observed). However, the other antigens such as B/Colorado and H1N1 (A/Brisbane), and to a lesser extent H3N2 (A/Kansas) and B/Phuket, exhibited degradation, loss of trimer, or loss of other key properties under these conditions. For example,
[0196] When the same purified antigens are conjugated to TMV, according to multiple embodiments and alternatives, the stability profile and storage potency changes. In some embodiments, the inventive method enhances a measure of stability of a conjugated compound comprising a protein and virus particle, and includes activating the virus particle and then mixing the virus particle and the antigen in a conjugation reaction to form a conjugate mixture, resulting in enhanced stability when the conjugated compound is placed in an unrefrigerated environment and after a time period of at least 42 days following a release date. An exemplary storage temperature is at least 20° C. The stability enhancement can be gauged by comparing the stability of the conjugate mixture to that of the antigen alone. A suitable measure is any one or more of antigen concentration, antigen integrity, or antigen potency. For example, when the measure of stability is antigen concentration, as measured by BCA or other appropriate methodology, a difference between concentration of the conjugated compound and concentration of the antigen alone of at least 10% is within the scope of present embodiments. Likewise, when the measure of stability is antigen integrity, as measured by SDS-PAGE, SEC-HPLC or other appropriate methodology, a difference between integrity of the conjugated compound and integrity of the antigen alone of at least 10% is within the scope of present embodiments. Likewise, when the measure of stability is antigen potency, as measured by antigen-antibody interaction based on ELISA results, or VaxArray, surface plasmon resonance or other appropriate methodology, a difference between potency of the conjugated compound and potency of the antigen alone of at least 30% is within the scope of present embodiments.
[0197] Accordingly, the following tables provide the stability data of several monovalent formulations (at a TMV to antigen ratio of 1:1) at release and various times after filling into vials and stored under refrigerated conditions (2° to 8° C.):
TABLE-US-00026 TABLE 24 Stability of the H1NI (A/Michigan) to TMV Conjugate Under Refrigerated Conditions Test Initial Test Parameters Method (CoA) 1 month 3 months 6 months Appearance Appearance Clear, Clear, Cloudy, Cloudy, Liquid Liquid Liquid Liquid Physical/Chemical pH 7.6 7.5 7.4 7.5 Properties Protein BCA 0.898 1.066 1.101 0.994 Concentration Purity SDS PAGE >99.0 94.3 90.7 91.7 Storage VaxArray 325 329 415 208 Potency Average Size DLS 85.8 98.0 64.2 97.8 Radius Polydispersity 53.9 54.2 54.3 55.2
TABLE-US-00027 TABLE 25 Stability of the H3N2 (A/Singapore) to TMV Conjugate Under Refrigerated Conditions Test Initial Test Parameters Method (CoA) 1 month 3 months 6 months Appearance Appearance Clear, Clear, Cloudy, Cloudy, Liquid Liquid Liquid Liquid Physical/Chemical pH 7.6 7.4 7.4 7.5 Properties Protein BCA 0.828 1.025 0.947 0.957 Concentration Purity SDS PAGE >99.0 94.9 92.8 92.9 Storage VaxArry 363 496 468 500 Potency Average Size DLS 72.1 86.3 77.8 71.1 Radius Polydispersity 43 52.6 38.7 35.4
TABLE-US-00028 TABLE 26 Stability of the B/Phuket to TMV Conjugate Under Refrigerated Conditions Test Initial Test Parameters Method (CoA) 1 month 3 months 6 months Appearance Appearance Cloudy, Cloudy, Cloudy, Cloudy, Liquid Liquid Liquid Liquid Physical/Chemical pH 7.6 7.5 7.4 7.5 Properties Protein BCA 0.874 1.010 0.995 0.940 Concentration Purity SDS PAGE >99.0 97.1 95.4 95.1 Storage VaxArry 333 393 442 477 Potency Average Size DLS 1040.7 1094.1 1428.2 1284.9 Radius Polydispersity 47.5 42.1 49.6 53.3
TABLE-US-00029 TABLE 27 Stability of the B/Colorado to TMV Conjugate Under Refrigerated Conditions Test Test Initial 1 3 6 Parameters Method (CoA) month months months Appear- Appear- Cloudy, Cloudy, Cloudy, Cloudy, ance ance Liquid Liquid Liquid Liquid Physical/ pH 7.6 7.5 7.5 7.5 Chemical Properties Protein BCA 0.961 1.020 1.077 0.959 Concen- tration Purity SDS >99.0 96.0 96.0 94.9 PAGE Storage VaxArry 218 653 599 585 Potency Average DLS 2377.8 1025.7 1337.6 1153.9 Size Radius Poly- 49.5 55.6 53.3 ≥57.1 dispersity
[0198] In each of the conjugates described in Tables 24-27, the purity, pH, protein concentration, and storage potency is maintained through at least six months of storage under refrigerated conditions. Further, the polydiversity is also consistent over this timeframe. Polydiversity refers to the variability of particle size in a complex product, and generally the lower the polydiversity than the better the product.
[0199] In addition to the monovalent formulations, the following quadrivalent conjugate produced according to multiple embodiments and alternatives at a 1:1 TMV to antigen ratio exhibits strong stability under both refrigerated (4° to 8° C.) and room temperature (22° to 28° C.) conditions'
TABLE-US-00030 TABLE 28 Stability of the Quadrivalent Conjugate Under Refrigerated Conditions Test Test Parameters Method Initial (CoA) 1 month 3 months 6 months Appearance Appearance Cloudy, Liquid Cloudy, Liquid Cloudy, Liquid Cloudy, Liquid Physical/ pH 7.5 7.5 7.4 7.5 Chemical Properties Protein BCA 0.799 0.911 0.983 0.953 Concentration Identity VaxArray Antigen Antigen Antigen Antigen Binding Binding Binding Binding Occurs Occurs Occurs Occurs Storage VaxArray A/Michigan: A/Michigan: A/Michigan: A/Michigan: Potency NtK = NtK = NtK = NtK = 123 μg/ml 155 μg/ml 103 μg/ml 123 μg/ml a/Singapore: a/Singapore: a/Singapore: a/Singapore: NtK = NtK = NtK = NtK = 106 μg/ml 110 μg/ml 106 μg/ml 101 μg/ml B/Phuket: B/Phuket: B/Phuket: B/Phuket: NtK = NtK = NtK = NtK = 117 μg/ml 140 μg/ml 114 μg/ml 116 μg/ml B/Colorado: B/Colorado: B/Colorado: B/Colorado: NtK = NtK = NtK = NtK = 78 μg/ml 179 μg/ml 134 μg/ml 134 μg/ml
TABLE-US-00031 TABLE 29A Stability of the Quadrivalent Conjugate Under Room Temperature Conditions Test Test Parameters Method Initial (CoA) 2 weeks 1 month 2 months Appearance Appearance Cloudy, Liquid Cloudy, Liquid Cloudy, Liquid Cloudy, Liquid Physical/ pH 7.5 7.5 7.5 7.4 Chemical Properties Protein BCA 0.799 0.959 0.909 1.098 Concentration Identity VaxArray Antigen Antigen Antigen Antigen Binding Binding Binding Binding Occurs Occurs Occurs Occurs Storage VaxArray A/Michigan: A/Michigan: A/Michigan: A/Michigan: Potency NtK = NtK = NtK = NtK = 123 μg/ml 115 μg/ml 126 μg/ml 24 μg/ml a/Singapore: a/Singapore: a/Singapore: a/Singapore: NtK = NtK = NtK = NtK = 106 μg/ml 108 μg/ml 173 μg/ml 29 μg/ml B/Phuket: B/Phuket: B/Phuket: B/Phuket: NtK = NtK = NtK = NtK = 117 μg/ml 96 μg/ml 84 μg/ml 29 μg/ml B/Colorado: B/Colorado: B/Colorado: B/Colorado: NtK = NtK = NtK = NtK = 78 μg/ml 62 μg/ml 124 μg/ml 26 μg/ml
TABLE-US-00032 TABLE 29B Stability of the Quadrivalent Conjugate Under Room Temperature Conditions Test Test Parameters Method Initial (CoA) 3 months 6 months Appearance Appearance Cloudy, Cloudy, Cloudy, Liquid Liquid Liquid Physical/ pH 7.5 7.4 7.5 Chemical Properties Protein BCA 0.799 0.980 0.920 Concentration Identity VaxArray Antigen Antigen Antigen Binding Binding Binding Occurs Occurs Occurs Storage VaxArray A/Michigan: A/Michigan: A/Michigan: Potency NtK = NtK = NtK = 123 μg/ml 113 μg/ml 114 μg/ml a/Singapore: a/Singapore: a/Singapore: NtK = NtK = NtK = 106 μg/ml 115 μg/ml 80 μg/ml B/Phuket: B/Phuket: B/Phuket: NtK = NtK = NtK = 117 μg/ml 80 μg/ml 99 μg/ml B/Colorado: B/Colorado: B/Colorado: NtK = NtK = NtK = 78 μg/ml 139 μg/ml 120 μg/ml
[0200] Tables 28, 29A and 29B illustrate that the quadrivalent conjugate remains consistent and stable in terms of protein concentration, storage potency, pH and appearance under both refrigerated and room temperature conditions for at least six months. Table 30 provides the percent change in the storage potency of the various antigens described in Tables 29A and 29B by comparing the initial potency to the storage potency at the particular time.
TABLE-US-00033 TABLE 30 Percent Change in Storage Potency from the Initial Potency via VaxArray 2 Weeks 1 month 2 months 3 months 6 months A/Michigan 93.50% 102.44% 19.51% 91.87% 92.68% A/Singapore 101.89% 163.21% 27.36% 108.49% 75.47% B/Phuket 82.05% 71.80% 24.79% 68.00% 84.62% B/Colorado 79.49% 158.97% 33.33% 178.00% 153.85%
[0201] Accordingly, as shown in Table 30, when the conjugate was placed in the unrefrigerated environment, the storage potency at the end of 30 days was at least 70% of the initial potency of the conjugate mixture within the first day post-conjugation. At the end of 90 days, the storage potency of the conjugate mixture stored in the unrefrigerated environment was at least 68% of the initial potency, and the storage potency of the conjugate mixture was at least 75% at the end of at least 180 days.
[0202] The following tables illustrate the stabilizing effect of the embodiments described herein by comparing the release conditions of the purified recombinant antigen with the same protein conjugated to TMV according to multiple embodiments and alternatives. Furthermore, stability after six months under refrigerated conditions (4° to 8° C.) was compared between the purified antigen and the same antigen conjugated to TMV by analyzing the protein concentration, potency, SDS-page purity, and PH, as follows:
TABLE-US-00034 TABLE 31 Comparison Between the Stability of Purified B/Colorado Antigen and the B/Colorado to TMV Conjugate Colorado Release Colorado 6 month Data Stability Free Conjugated Free Conjugated Assay Antigen (1:1) Antigen (1:1) BCA (mg/mL) 0.848 0.961 0.777 0.959 VaxArray Potency 541 218 1082 585 (μg/mL) SDS PAGE Purity (%) 99 >99.0 35 94.9 pH 7.3 7.6 7.4 7.5
TABLE-US-00035 TABLE 32 Comparison Between the Stability of Purified B/Phuket Antigen and the B/Phuket to TMV Conjugate Phuket Release Phuket 6 month Data Stability Free Conjugated Free Conjugated Assay Antigen (1:1) Antigen (1:1) BCA (mg/mL) 0.957 0.874 0.819 0.940 VaxArray Potency 910 333 924 447 (μg/mL) SDS PAGE Purity (%) 96.1 >99.0 91.0 95.1 pH 7.4 7.6 7.4 7.5
TABLE-US-00036 TABLE 33 Comparison Between the Stability of Purified H3N2 (A/Singapore) Antigen and the H3N2 (A/Singapore) to TMV Conjugate Singapore Release Singapore 6 month Data Stability Free Conjugated Free Conjugated Assay Antigen (1:1) Antigen (1:1) BCA (mg/mL) 0.855 0.828 0.795 0.957 VaxArray Potency 746 363 1089 500 (μg/mL) SDS PAGE Purity (%) >99 >99.0 >99 92.9 pH 7.4 7.6 7.3 7.5
TABLE-US-00037 TABLE 34 Comparison Between the Stability of Purified H1NI (A/Michigan) Antigen and the H1NI (A/Michigan) to TMV Conjugate Michigan Release Michigan 6 month Data Stability Free Conjugated Free Conjugated Assay Antigen (1:1) Antigen (1:1) BCA (mg/mL) 1.081 0.898 0.921 0.994 VaxArray Potency 93 325 1176 208 (μg/mL) SDS PAGE Purity (%) 97 >99.0 76 91.7 pH 7.4 7.6 7.3 7.5
[0203] Tables 31-34 illustrate the stability inducing properties of the purification and conjugation embodiments, most clearly for the B/Colorado, B/Phuket, and H1N1 (A/Michigan) antigens in terms of purity measures. For the H3N2 (A/Singapore) and B/Colorado antigens, the stability of the conjugate is also shown in terms of antigen concentration. As shown in Tables 31-34, the purification and conjugation processes, according to multiple embodiments and alternatives, stabilized the antigen's physical properties, antigenic reactivity and other quantitative stability features.
[0204] Furthermore, Tables 29A, 29B, and 30 illustrate that the quadrivalent conjugate, produced according to multiple embodiments and alternatives, exhibits strong stability measures for at least six months, or twenty-four weeks, at room temperature storage (22° to 28° C.). Compared to conventional vaccines which exhibit an average stability of ˜5 weeks at room temperature (as discussed in the F. Coenen article mentioned above), the vaccines according to multiple embodiments and alternatives exhibit stability for at least 5× greater than conventional influenza vaccines and several times longer than purified antigens. Accordingly, the formulation and conjugation processes according to multiple embodiments and alternatives stabilize extremely unstable antigens—such as B/Colorado—and extend the stability of other antigens—such as H3N2 (A/Singapore), H1N1 (A/Michigan), and B/Phuket—far beyond the stability limits of free-antigens and conventional vaccines.
[0205] Another embodiment, referred to herein as embodiment A, and being a method of use, comprises administering to a subject a compound manufactured by conjugating a protein and a virus particle, i.e., activating the virus particle, then mixing the virus particle and the protein in a conjugation reaction to form a conjugate mixture, wherein when placed in an unrefrigerated environment at a storage temperature for a time period, an integrity or a concentration of the conjugate mixture is at least 90% of an initial integrity or an initial concentration of the conjugate mixture, wherein the time period is at least 42 days after a release date of the conjugate mixture. The subject may be a human being. An exemplary storage temperature is at least 20° C.
[0206] In an embodiment within the scope of embodiment A, and referred to herein as embodiment B, activating the virus particle comprises exposing the virus particle to a conjugation environment at a pH of about 5.5 or less. In an embodiment within the scope of embodiment A, and referred to herein as embodiment C, the virus particle is an enveloped virus. In an embodiment within the scope embodiment A, and referred to herein as embodiment D, the protein is an antigen. In an embodiment within the scope of embodiment A, and referred to herein as embodiment E, the antigen is hemagglutinin antigen. In an embodiment within the scope of embodiment A, and referred to herein as embodiment F, the time period is at least 90 days after the release date of the conjugate mixture. In an embodiment within the scope of embodiment A, and referred to herein as embodiment G, the time period is at least 180 days after the release date of the conjugate mixture. Accordingly, a method of use is described herein in which the vaccine described in connection with embodiment A is administered to a subject. This method may be further defined by incorporating the additional features of any one or more of embodiments B, C, D, E, F, or G.
[0207] Another embodiment, referred to herein as embodiment H, and being a method of use, comprises administering to a subject a vaccine manufactured by conjugating a protein and a virus, i.e., activating the virus, then mixing the virus and the protein in a conjugation reaction to form a conjugate mixture, wherein when placed in an unrefrigerated environment at a storage temperature for a time period, an integrity or a concentration of the conjugate mixture is at least 90% of an initial integrity or an initial concentration of the conjugate mixture, wherein the time period is at least 42 days after a release date of the conjugate mixture. The subject may be a human being. An exemplary storage temperature is at least 20° C.
[0208] In an embodiment within the scope of embodiment H, and referred to herein as embodiment I, activating the virus comprises exposing the virus to a conjugation environment at a pH of about 5.5 or less. In an embodiment within the scope of embodiment H, and referred to herein as embodiment I, the virus is tobacco mosaic virus. In an embodiment within the scope of embodiment H, and referred to herein as embodiment J, the protein is an antigen. In an embodiment within the scope of embodiment H, and referred to herein as embodiment K, the antigen is hemagglutinin antigen. In an embodiment within the scope of embodiment H, and referred to herein as embodiment L, the time period is at least 90 days after the release date of the conjugate mixture. In an embodiment within the scope of embodiment H, and referred to herein as embodiment M, the time period is at least 180 days after the release date of the conjugate mixture. Accordingly, a method of use is described herein in which the vaccine described in connection with embodiment H is administered to a subject. This method may be further defined by incorporating the additional features of any one or more of embodiments I, J, K, L, or M.
[0209] Another embodiment, referred to herein as embodiment N, and being a method for enhancing a measure of stability of a conjugated compound comprising a protein and a virus particle, the method comprising activating the virus particle, and then mixing the virus particle and the protein in a conjugation reaction to form a conjugate mixture, wherein when placed in an unrefrigerated environment at a storage temperature for a time period, an integrity or a concentration of the conjugate mixture is at least 90% of an initial integrity or an initial concentration of the conjugate mixture, wherein the time period is at least 42 days after a release date of the conjugate mixture. An exemplary storage temperature is at least 20° C. In some embodiments, activating the virus particle comprises exposing the virus particle to a conjugation environment at a pH of about 5.5 or less.
[0210] In an embodiment within the scope of embodiment N, and referred to herein as embodiment O, the virus particle is an enveloped virus. In an embodiment within the scope of embodiment N, and referred to herein as embodiment P, the protein is an antigen. In an embodiment within the scope of embodiment N, and referred to herein as embodiment Q, the antigen is hemagglutinin antigen. In an embodiment within the scope of embodiment N, and referred to herein as embodiment R, the time period is at least 90 days after the release date of the conjugate mixture. In an embodiment within the scope of embodiment N, and referred to herein as embodiment S, the time period is at least 180 days after the release date of the conjugate mixture. This method may be further defined by incorporating the additional features of any one or more of embodiments O, P, Q, R or S.
[0211] Another embodiment, referred to herein as embodiment T, and being a method for enhancing a measure of stability of a conjugated compound comprising a protein and a virus, the method comprising activating the virus, then mixing the virus and the protein in a conjugation reaction to form a conjugate mixture, wherein when placed in an unrefrigerated environment at a storage temperature for a time period, an integrity or a concentration of the conjugate mixture is at least 90% of an initial integrity or an initial concentration of the conjugate mixture, wherein the time period is at least 42 days after a release date of the conjugate mixture. An exemplary storage temperature is at least 20° C. In some embodiments, activating the virus comprises exposing the virus particle to a conjugation environment at a pH of about 5.5 or less.
[0212] In an embodiment within the scope of embodiment T, and referred to herein as embodiment U, the virus is tobacco mosaic virus. In an embodiment within the scope of embodiment T, and referred to herein as embodiment V, the protein is an antigen. In an embodiment within the scope of embodiment T, and referred to herein as embodiment W, the antigen is hemagglutinin antigen. In an embodiment within the scope of embodiment T, and referred to herein as embodiment X, the time period is at least 90 days after the release date of the conjugate mixture. In an embodiment within the scope of embodiment T, and referred to herein as embodiment Y, the time period is at least 180 days after the release date of the conjugate mixture. This method may be further defined by incorporating the additional features of any one or more of embodiments U, V, W, X, or Y.
[0213] Another embodiment, referred to herein as embodiment Z, and being a chemical compound, comprises a conjugated protein and a virus particle wherein the protein is chemically associated with lysine residues on a surface of the virus, and wherein when the chemical compound is placed in an unrefrigerated environment at a storage temperature for a time period, an integrity or a concentration of the chemical compound at the end of the time period is at least 90% of an initial integrity or an initial concentration of the chemical compound, wherein the time period is at least 42 days a release date of the chemical compound. An exemplary storage temperature is at least 20° C.
[0214] In an embodiment within the scope of embodiment Z, and referred to herein as embodiment AA, the virus particle is a virus. In an embodiment within the scope of embodiment Z, and referred to herein as embodiment BB, the virus is an enveloped virus. In an embodiment within the scope of embodiment Z, and referred to herein as embodiment CC, the virus is a tobacco mosaic virus. In an embodiment within the scope of embodiment Z, and referred to herein as embodiment DD, the time period is at least 90 days after the release date of the conjugate mixture. In an embodiment within the scope of embodiment Z, and referred to herein as embodiment EE, the time period is at least 180 days after the release date of the conjugate mixture. This compound may be further defined by incorporating the additional features of any one or more of embodiments AA, BB, CC, DD or EE.
[0215] Another embodiment, referred to herein as embodiment FF, and being a method for enhancing a measure of stability of a conjugated compound comprising a protein and a virus particle, the method comprising activating the virus particle and then mixing the virus particle and an antigen in a conjugation reaction to form a conjugate mixture, wherein when placed in an unrefrigerated environment at a storage temperature and after a time period of at least 42 days following a release date of the conjugate mixture, the conjugate mixture demonstrates a stability that exceeds an initial stability of the conjugate mixture stability for the antigen alone as measured by one or more of antigen concentration, antigen integrity, or antigen potency. An exemplary storage temperature is at least 20° C. In some embodiments, activating the virus comprises exposing the virus particle to a conjugation environment at a pH of about 5.5 or less.
[0216] In an embodiment within the scope of embodiment FF, and being referred to herein as embodiment GG, the antigen is hemagglutinin antigen. In an embodiment within the scope of embodiment FF, and referred to herein as embodiment HH, the virus is tobacco mosaic virus. In an embodiment within the scope of embodiment FF, and referred to herein as embodiment II, the time period is at least 90 days after the release date of the conjugate mixture. In an embodiment within the scope of FF, and referred to herein as embodiment JJ, the time period is at least 180 days after the release date of the conjugate mixture. In an embodiment within the scope of FF, and referred to herein as embodiment KK, the measure of stability is antigen concentration, and a difference between concentration of the conjugate mixture and concentration of the antigen alone is at least 10%. In an embodiment within the scope of FF, and referred to herein as embodiment LL, the measure of stability is antigen integrity, and a difference between integrity of the conjugate mixture and integrity of the antigen alone is at least 10%. In an embodiment within the scope of FF, and referred to herein as embodiment MM, the measure of stability is antigen potency, and a difference between potency of the conjugate mixture and potency of the antigen alone is at least 10%. In an embodiment within the scope of FF, and referred to herein as embodiment NN, the measure of stability is antigen potency, and a storage potency of the conjugate mixture at the end of the time period is at least 70% of an initial potency of the conjugate mixture. This method may be further defined by incorporating the additional features of any one or more of embodiments GG, HH, II, JJ, KK, LL, MM or NN.
[0217] It will be understood that the embodiments described herein are not limited in their application to the details of the teachings and descriptions set forth, or as illustrated in the accompanying figures. Rather, it will be understood that the present embodiments and alternatives, as described and claimed herein, are capable of being practiced or carried out in various ways. Also, it is to be understood that words and phrases used herein are for the purpose of description and should not be regarded as limiting. The use herein of “including,” “comprising,” “e.g.,” “containing,” or “having” and variations of those words is meant to encompass the items listed thereafter, and equivalents of those, as well as additional items.
[0218] Accordingly, the foregoing descriptions of several embodiments and alternatives are meant to illustrate, rather than to serve as limits on the scope of what has been disclosed herein. The descriptions herein are not intended to be exhaustive, nor are they meant to limit the understanding of the embodiments to the precise forms disclosed. It will be understood by those having ordinary skill in the art that modifications and variations of these embodiments are reasonably possible in light of the above teachings and descriptions.