Multivalent stable vaccine composition and methods of making same
10293041 ยท 2019-05-21
Assignee
Inventors
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
A61K39/39
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
A61K39/39
HUMAN NECESSITIES
Abstract
Stable immunogenic composition capable of eliciting a robust and durable immune response yielding a measurable increase in neutralizing antibodies at least 200 days post-administration, comprising at least one antigen consisting of a ribosome inactivating protein and at least one antigen comprising a toxin derived from bacterial spores. Method making and using a stable immunogenic composition capable of eliciting a stable immune response yielding a measurable increase in neutralizing antibodies at least 200 days post-administration, comprising providing an immunogenic composition comprising at least one antigen comprising a ribosome inactivating protein and at least one antigen comprising a toxin derived from bacterial spores and administering the immunogenic composition to an individual.
Claims
1. An immunogenic composition comprising a first antigen comprising a ribosome inactivating protein and a second antigen comprising a toxin derived from bacterial spores, wherein the immunogenic composition is formulated to elicit protective immunity to each of the first and second antigenic components without causing immune interference between the first and the second antigens after administration to an individual, further comprising at least one disaccharide glass forming excipient, aluminum hydroxide, an effective amount of glycopyranoside lipid A, and a buffer consisting of 50% glycerol buffer and 50% histidine buffer.
2. The composition of claim 1, wherein the composition yields a measurable increase in neutralizing antibodies to the first antigen comprising the ribosome inactivating protein.
3. The composition of claim 1, wherein the composition is capable of yielding a measurable increase in neutralizing antibodies to the second antigen comprising a toxin derived from bacterial spores.
4. The composition of claim 1, wherein the composition is capable of yielding a measurable increase in neutralizing antibodies to both the first and the second antigen.
5. The composition of claim 1, wherein the composition elicits a measurable increase in neutralizing antibodies to the first antigen at least 200 days post-administration.
6. The composition of claim 1, wherein the composition elicits a measurable increase in neutralizing antibodies to the second antigen at least 200 days post-administration.
7. The composition of claim 1, wherein the composition elicits a measurable increase in neutralizing antibodies to the first and the second antigen at least 200 days post-administration.
8. The composition of claim 1, further wherein the composition is administered prior to exposure to a toxin or infectious agent.
9. The composition of claim 1, further wherein the composition is administered after exposure to a toxin or infectious agent.
10. The composition of claim 1, further comprising at least one adjuvant selected from the group consisting of aluminum salts, water-in-oil emulsions, oil-in-water emulsions, self-assembling macrostructures, cytokines, saponins, toll-like receptor agonists, immunostimulatory double stranded RNA species, unmethylated DNA oligonucleotides, and polymeric microparticles and nanostructures.
11. The composition of claim 1, further comprising at least one adjuvant comprising bacterial DNA and another adjuvant comprising flagellin.
12. The composition of claim 1, further comprising monophosphoryl lipid A.
13. The composition of claim 1, further comprising a co-adjuvant system consisting essentially of an aluminum salt, monophosphoryl lipid A, QS-21 and CpG sequences.
14. The composition of claim 1, wherein the first antigen is derived from ricin toxin and the second antigen is derived from Bacillus anthracis.
15. The composition of claim 14, wherein the first antigen derived from ricin toxin consists of a purified ricin A chain.
16. The composition of claim 14, wherein the second antigen derived from Bacillus anthracis consists of protective antigen (PA).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(7) The present invention is directed towards a combination vaccine that comprises vaccine components that are suitable for the prevention, amelioration and treatment of exposure to toxins consisting of ribosome inactivating proteins and concurrently exposure to toxins elaborated by spore forming bacteria. The vaccine of the present invention elicits protective immunity to each of the antigenic components without causing immune interference after administration to an individual. In a preferred embodiment, the combination vaccine is administered prior to exposure to the toxin or infectious agent. In another aspect of the invention, the vaccine can be administered after exposure to the toxin or infectious agent. Further, in another embodiment of the invention, the vaccine can be administered after exposure to the infectious agent or toxin in combination with therapies intended to cure or ameliorate the effects of the toxins that are responsible for the morbidity of disease exposure.
(8) In another embodiment of the current invention, the combination vaccine is formulated with adjuvants that preferentially induce neutralizing antibodies against the corresponding antigenic vaccine ingredients.
(9) In a preferred embodiment of the current invention, the combination vaccine is formulated with antigen derived from ricin toxin, consisting of the purified ricin A chain combined with antigen consisting of the protective antigen (PA) derived from Bacillus anthracis.
(10) The advantages of the present invention include a combination vaccine which can confer protection against exposure to toxins of that are secreted by Bacillus anthracis during infection and exposure to ribosome inactivating proteins.
(11) The vaccine of the invention provides protective immune responses to toxins with no interference or immune competition among the antigens that are present in the vaccine. Thus, a single shot will confer immunogenicity simultaneously in a single administration against anthrax diseases and exposure to ricin toxin. The vaccine is easier to administer in fewer injections to achieve protective and long lasting immunity to ribosomal inactivating toxins and the toxins causing pathology against anthrax disease upon exposure to spores of Bacillus anthracis. Since a single shot would afford immunity against anthrax and ribosome inactivating proteins, the cost of vaccination would be reduced. The vaccine of the present invention would be advantageous by reducing the number of visits required for full vaccination and the numbers of vaccine administrations necessary to achieve full protection. Thus, the present invention provides a vaccine that is more acceptable for rapid onset of protective immunity.
(12) The following examples illustrate the various embodiments of the present invention and are not meant to be limiting in scope based on such examples.
Example I: Production of Vaccine Antigen Ricin a Chain
(13) Ricin A chain is structurally unstable, with improvements being necessary to achieve the objective of long lasting and rapid onset immunity using 2 vaccine doses or fewer. The ricin A chain is extremely labile in aqueous buffers without stabilizers, leading to unfolding and aggregation of the protein in solution. Protein unfolding also occurs on the surface of aluminum adjuvant particles in the liquid suspension vaccines. The summation of the studies with liquid aluminum-adsorbed vaccine in mice, rabbits, humans and macaques indicate that improvements will be necessary to achieve the objective of long lasting and rapid onset immunity using two vaccine doses or fewer. Because the effectiveness of ricin A chain vaccine is thought to be associated with protein configuration, as the majority of neutralizing antibodies recognize conformational determinants, efforts were initiated to stabilize RiVax bound to conventional aluminum adjuvant by lyophilization. During lyophilization, aggregation of colloidal aluminum hydroxide suspensions can be inhibited by reducing the extent of their freeze-concentration by using formulations that contain high concentrations of glass-forming excipients, and by limiting the time over which the freeze-concentrated suspensions can aggregate by using rapid cooling procedures to maximize the kinetics of glass formation (Clausi, Cummiskey, et al., 2008, Influence of particle size and antigen binding on effectiveness of aluminum salt adjuvants in a model lysozyme vaccine, J Pharm Sci, v 97:5252-62; Clausi, Merkley, et al., 2008, Inhibition of aggregation of aluminum hydroxide adjuvant during freezing and drying, J Pharm Sci, v 97:2049-61). This has been usually accomplished by freeze drying in the presence of disaccharides such as trehalose and other excipients that promote a glass state during process and storage. Proteins can be stored for long-term as long as the product is stored below its glass transition temperature (T.sub.g), above which the material transitions into a rubbery state.
(14) A small scale process for purifying RiVax was developed and implemented for initial human Phase 1 trials (Smallshaw, Richardson, et al., 2005, Preclinical toxicity and efficacy testing of RiVax, a recombinant protein vaccine against ricin, Vaccine, v 23:4775-84; Vitetta, Smallshaw, et al., 2006, A pilot clinical trial of a recombinant ricin vaccine in normal humans, Proceedings of the National Academy of Sciences of the United States of America, v 103:2268-73). During these initial small Phase 1 studies in humans, the vaccine purification process was improved and scaled up to increase protein yields and to develop an aluminum-adsorbed product. In the process of developing the RiVax vaccine candidate, the bulk drug substance was stabilized in aqueous buffer by the inclusion of 50% glycerol (Peek, Brey, et al., 2007, A rapid, three-step process for the preformulation of a recombinant ricin toxin A-chain vaccine, Journal of Pharmaceutical Sciences, v 96:44-60) which was removed by the final process to make vaccine adsorbed to Alhydrogel. Because of the potential impact of destabilization in liquid buffers and the potential importance of conformational determinants, a major effort has been undertaken to stabilize RiVax adsorbed to Alhydrogel.
(15) A robust and scalable process was developed for the purification of RiVax, a ricin A chain mutant vaccine candidate. The purified RiVax is stored in 50% glycerol for further formulation development and processing in the generation of RiVax, AIOH adsorbed or RiVax-TR, (aluminum-adsorbed, lyophilized for reconstitution). RiVax is stored in glycerol at 20 C., and is subject to dialysis or ultrafiltration to remove glycerol before adsorption to AIOH and further processing and vial filling. The rationale for the use of glycerol concerns identifying conditions for the retention of RiVax structure during storage prior to aluminum adsorption under conditions in which the protein does not significantly aggregate or unfold. These conditions were identified in extensive screens of conditions to examine tertiary conformation, secondary protein structure aggregation and the influence of generally regarded as safe (GRAS) excipients on possible unfolding events. Through screens, it was found that the most efficient stabilizer was glycerol for the retention of native structure. It may be possible to further scale up runs to eliminate the glycerol holding/storage step in favor of direct absorption to AlOH, which may stabilize protein structure and immunogenicity.
(16) A recombinant E. coli process was developed for the manufacture of RiVax according to principles of rational design. This process is suitable for implementation within a GMP manufacturing environment and has been implemented in 7, 100 liter (L) runs, 4 of which were conducted as process development runs to scale up from 10 L, and 2 were conducted in a cGMP environment as engineering or demonstration runs, and one run was conducted under cGMP. All runs were conducted at the Cambrex/Lonza facility in Baltimore, Md. Material generated from one of the process development runs has been established as a reference protein, and material generated from one of the engineering runs has been used extensively in adjuvant formulation characterization
(17) Strain construction and fermentation development proceeded according to the following protocol: Initially, the coding sequence for the mature RiVax protein with mutations Y80A/V76M was cloned into the pET28a vector and transformed and stored in the E. coli strain BL21(DE3) at the University of Texas Southwestern Medical Center (UTSWMC). The product was expressed in recombinant E. coli BL21(DE3) with low yields, approximately 6 mg/L. The host strain BL21(DE3) can shed phage particles under certain stress conditions and is thus not suitable for cGMP manufacturing at large scale in multi-use facilities. The pET28a-Y80A/V76M construct was transformed into BLR(DE3) and HMS174(DE3), which are recA- and do not shed phage particles, and evaluated for product expression. Eight colonies from each transformation were streaked onto LB Kan agar and four well-isolated colonies from each transformation were grown out in 0.5 Vegan TB with 50 mg/L kanamycin for IPTG-inducible RiVax expression analysis by SDS-PAGE. Post-induction samples were taken 3.0 to 3.5 hours after the addition of IPTG and samples were analyzed by SDS-PAGE for product expression. A protein of approximately 30 kDa was clearly induced in each of these cultures. The insert sequence from both the BLR 3 and HMS174 A clones was 100% homologous with the predicted sequence.
(18) Fermentation development and optimization was conducted with the goals of increasing the cell mass and the fermentation titer of RiVax, as well as developing scalable methods for RiVax recovery in a soluble form using cGMP compliant medium and process. Fermentations were performed in B. Braun fermenters at the 10 L scale in batch and fed-batch mode with varying inducer (IPTG) concentration. The main variables that were examined included 1) sources of peptone from non-animal origin 2) concentration of IPTG inducer and 3) carbon source. Batch fermentations were performed for one clone from each of the E. coli host strains: Clone BLR 3 and Clone HMS A. Starter cultures of ECPM medium (25 mL) were inoculated with 0.25 mL of either BLR 3 or HMS A frozen stock culture and grown 10 hours at 37 C., 200 RPM in a gyratory shaker. These cultures were used to inoculate 200 mL of Batch Fermentor Medium in 1000 mL baffled shake flasks. The 200 mL cultures were allowed to grow at 37 C., 200 RPM for nine hours then used to inoculate 10 L of Batch Fermentation Medium in 14 L BIOFLO 3000 vessels (New Brunswick Scientific). Casamino Acids and other non-animal origin nitrogen sources were screened for production of cell mass with the result that yeast extract was capable of supporting high levels of cell mass. Initial expression studies revealed that a low level of uninduced expression in glycerol-grown BLR 3 fermentations, and stricter dependence of expression upon IPTG induction in clones of HMS174. Consequently, HMS174 was selected for further development. Results of ensuing studies indicated that the bulk of the IPTG-induced expression was located in inclusion bodies (the insoluble fraction). A large fraction of the RiVax remained insoluble after lysis in the presence of 3 M urea at pH 7.1. In subsequent experiments, the inducer concentration was reduced to 0.75 mM and 0.25 mM from 1.0 mM. By reducing the IPTG concentration, a significant portion of the expressed protein was in the soluble fraction. The yields in the soluble fraction as measured on a cation exchange column ranged between 1.2 g/L and 1.8 g/L in the soluble fraction.
(19) The purification process comprises capture chromatography using cation exchange resin, endotoxin removal using a charged filter followed by hydrophobic interaction chromatography (HIC) and finally ultrafiltration/diafiltration (UF/DF) to affect buffer exchange in the final formulation for storage of bulk protein. The processing steps include Poros HS50 Chromatography, Endotoxin Removal by Mustang E Filtration, Butyl-S Sepharose 6 FF Chromatography and Final Ultrafiltration/Diafiltration to obtain the final drug substance in 50% w/w glycerol.
(20) Three 100 L fermentations were executed to document this process for process development, one of them as a non-clinical GLP process. These batches were labeled 100 L Run #1 (batch 190-100L-SS-0511-05), 100 L Run #2 (batch 190-1001-FF-0801-05) and the GLP batch (190-100-GLP-FF-090105). Essentially the same results were obtained as with the 10 L fermentations, with slightly lower yields of protein in the soluble fraction. These runs were conducted as process development runs, prior to transferring the methodology to manufacturing, which was carried out in a separate facility.
(21) From analysis of the reverse phase and size exclusion HPLC data and spectral data from protein obtained from 100 L process development runs stored in 50% glycerol at 20 C. for 3 months, it was concluded that the protein stored under these conditions retained native configuration, and there was no evidence of degradation of protein profiles using two different HPLC methods (reverse phase and size exclusion HPLC) or SDS gel electrophoresis. Consequently, three subsequent lots were produced: two engineering run lots and one cGMP lot at the 100 L fermentation scale.
(22) The tangential flow filtrate (TFF) permeate from cell lysis was chromatographed with Poros HS50. Poros HS50 chromatography was performed using a K40 chromatography skid 4. For lot 190-0306-005 the elution product was collected from 0.1 OD A280 on the ascending slope to 0.06 OD A.sub.280 on the descending slope of the peak. The product from the Poros HS column was pumped through a Pall Mustang E filter to remove endotoxin. The Mustang filtered pool was stored for 2 hours at 2-8 C. prior to the Butyl S chromatography. The filtered Poros HS50 pool was conditioned by the addition of 3.6 L of 25 mM sodium phosphate, 0.6 M NaCl, 3.0 M Ammonium Sulfate, pH 6.5 and 2.5 L of 25 mM sodium phosphate, 4.0 M NaCl, 0.5 M Ammonium Sulfate, pH 6.5. The conditioned Butyl load had a conductivity of 151 mS/cm. The Butyl S purification was performed using a K40 chromatography skid. For lot 190-0306-005, the pre-elution eluate from 0.1 OD A.sub.280 to 0.725 OD A.sub.280 was collected as I L fractions in 2 L PETG bottles. The eluate, from 0.725 OD A.sub.280 on the ascending slope to 0.03 OD A.sub.280 on the descending slope, was collected in the elution media bag. Following the Butyl chromatography, the six fractions were combined with the elution product pool, total=11.4 L. The combined product was diluted 1:1 with formulation buffer (17 mM Histidine, 238 mM NaCl, 15% Glycerol, pH 6.0). The concentration/diafiltration was performed immediately following the dilution. The cGMP batch was released for clinical evaluation and final formulation with Alhydrogel adjuvant. In summary, a robust and scalable process with an overall yield of approximately 3-5 g of purified protein from 100 L fermentations, with reproducible batch yield. The fermentation yield has been calculated to be in the order of 700-1000 mgs of soluble protein per L of lysate.
Example II: Assessment of RiVax Stability Over Time
(23) A formal stability study was conducted over two years on RiVax protein from a former engineering run. The assay methodology and criteria for stability were based on the initial set of release specs established for the bulk protein during the first cGMP runs, Characterization tests have included generation of fluorescence spectra on the bulk protein, circular dichroism with thermal melt data, electrospray mass spectrometry, and N-terminal peptide sequencing. CD and fluorescence spectra are being more generally used for investigation into the process of monitoring protein configuration. The evaluation of stability was conducted at 2-4 C., 4 C., and 40. For protein stored at 20 C. or 2-4 C., there is little evidence of structural change, or the appearance of alternate species over two years (Table 1). A double minima (208 and 222 nm) was observed in the CD spectra of the 20 and 2-8 C. stability samples buffer at all times tested up to 24 months, suggesting retention of significant -helical character characteristic of T=0 (data not shown). The 40 C. sample displays virtually no negative ellipticity (data not shown). Sampling after the termination of the formal stability study indicated little evidence of gross structural changes. MALDI TOF analysis conducted in 2012 indicated little evidence of deamidated or oxidized peptide species.
(24) TABLE-US-00001 TABLE 1 24 months stability of RTA (reference batch) in 50% glycerol at 20 C. TIME (months) Assay 0 1 2 3 6 9 12 18 24 Protein concentration, mg/mL O.D. 280 0.322 0.287 0.316 0.353 0.303 0.307 0.304 0.330 0.330 Purity RP HPLC Major peak area 100 107 105 106 106 94 89 99 103 (% of T = 0) major peak, % 96 96 94 97 98 99 99 85 91.5 minor peak, % 4 4 6 3 2 1 1 15 8.5 SEC HPLC major peak, 100 100 100 100 100 100 100 100 100 % SDS Gel 100 100 100 100 100 100 100 100 100 Secondary structure CD, min peak, nm 208 208 208 208 208 208 208 208 208 Tertiary Structure Tm, C. 55 55 55 55 55 55 55 55 55 trp- peak, nm 326 326 326 326 326 326 327 327 327
(25) These studies indicate that RiVax protein in solution is stable for years in aqueous buffer in the presence of 50% glycerol. Consequently, in the new manufacturing campaign, we plan to retain the terminal step to add 50% glycerol to the bulk drug substance, with storage at 20 C., which will be used in subsequent formulations steps.
Example III: Stability of RiVax Adsorbed to Aluminum Hydroxide Adjuvant
(26) Studies were initiated to monitor the tertiary structure of RiVax on the surface of Aihydrogel using a fluorescence emission detection method that detects changes in tryptophan peak emission reflecting a change in the local environment of the residues from which water is excluded resulting in a red or blue shift indicative of a change in configuration in that region of the molecule.
(27) Measurable fluorescence spectra can be detected in vaccine prepared with concentrations of RiVax in excess of approximately 50 g/mL with 0.85 mgs of AlOH. Kinetics of the movement of the peak emission while the protein is adsorbed to Aihydrogel can be determined using a front face triangular geometry cuvette system. This system is a method to monitor changes of protein configuration under a variety of conditions. For example, the following data have been generated for a reference batch of vaccine stored at 2-4 C. and 40 C. (
Example IV: Manufacture of the Dominant Negative Inhibitor (DNI) for Pre- and Post-Exposure Vaccination
(28) The DNI protein was developed as a post-exposure therapeutic. A purification process was developed and cGMP lots were manufactured at the 300-600 Liter fermentation scale. These runs resulted in hundreds of grams of protein that were formulated with excipients to stabilize the protein prior to vaccine formulation and combination.
(29) The protective antigen (PA) gene of Bacillus anthracis was cloned into E. coli BL21 (DE3) using a pET22-b(+) vector (Novagen, Inc), and mutated by site directed mutagenesis. The resulting double mutant gene (K397D, D425K) encodes the DNI protein and resides on a 2.3 kb Nde I-Xho I fragment. The 5 end of this fragment contains the 23 amino acid pelB leader sequence, including its cleavage signal, which directs the secretion of the DNI protein to the periplasm. A ten amino acid N-terminal extension lies between the pelB signal cleavage site and a GAA codon (glu) that marks position #1 of the mature 83 kD PA as excreted by B. anthracis. This ten amino extension is a cloning artifact derived from pET22 vector sequence and does not affect the function of the active DNI protein since it is located on the 20 kb fragment that is cleaved at the cell surface. Two in-frame STOP codons, TAA TGA, terminate transcription immediately upstream of the 6His-Tag encoded by the pET22-b(+) vector. The original expression plasmid carried the bla gene encoding resistance to ampicillin. Therefore, this gene construct was recloned into pET24-based vectors encoding the NPT II gene for resistance to kanamycin for periplasmic and cytoplasmic expression of DNI. The DNI gene insert was recovered from the original DNI gene construct from Harvard University Medical School by digestion with Nde I and Xho I. Transformants were characterized by simple restriction analysis and used to purify sufficient plasmid DNA to isolate the Nde I-Xho I fragment containing the DNI gene. In addition to the DNI coding region, this fragment contained the pelB leader sequence and the 10 amino acid N-terminal extension. The Nde I-Xho I fragment was ligated into a pET 24 vector cut with Nde I and Xho I, and the ligation products used to transform E. coli BLR (DE3) to kanamycin resistance. Transformants were characterized by simple restriction analysis. The complete amino acid sequence of the DNI protein is shown in Table 2.
(30) TABLE-US-00002 TABLE2 AminoacidsequenceofDNIprotein MDIGINSDPMEVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTT GDLSIPSSELENIPSENQYFQSAIWSGFIKVKKSDEYTFATSADNHVTMW VDDQEVINKASNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTDSQ NKKEVISSDNLQLPELKQKSSNSRKKRSTSAGPTVPDRDNDGIPDSLEVE GYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVT GRIDKNVSPEARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTIS KNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSL AGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQT LATIKADENQLSQILAPNNYYPSKNLAPIALNAQKDFSSTPITMNYNQFL ELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTAR IIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNL QYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNIL IRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILS GYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYIS NPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG FIG. 13. Amino acid sequence of DNI PA83 with the locations of the substitutions at 397 (K to D) and 425 (D to K) also noted.
(31) Competent E. coli BLR (DE3) cells were obtained from Novagen, Inc. Ligation mixtures were used to transform competent E. coli BLR (DE3) cells to construct the recombinant DNI production strain following the procedures described by the manufacturer (Novagen, Inc.). Clones were selected for expression of the correct size protein band on SDS-PAGE gels with Coomassie staining. The fermentation and purification parameters were developed following standard processes. Both batch and fed-batch parameters were analyzed as were combinations of purification resins and combinations in order to maximize yield while maintaining a high level of purity and a low level of endotoxin, host cell protein (HCP) and DNA contamination. The DNI Drug Substance is an 83 kd protein consisting of 745 amino acids. There are no disulfide bonds present in the full length protein. The full length DNI protein is not capable of having a quaternary structure. However, once DNI is cleaved by furin, the resulting 63 kd protein forms either monomeric heptamers or hetero-heptamers with wt PA. The DNI protein is expressed in the periplasm of E coli after induction with IPTG. The product is susceptible to degradation by a neutral protease and the inclusion of EDTA in the buffers during processing seems to limit proteolysis. The cells are cultured in a medium that contains glycerol as a carbon source and Casamino Acids as the principal complex nitrogen source. A completely vegan carbon source was initially used, but a significant amount of uninduced product expression was seen with these reagents. The use of Casamino Acids as the complex nitrogen source prohibits leaky expression of the product prior to induction and results in extracts with limited DNI degradation. The fermentation is performed in a fed-batch mode with a simple linear feeding protocol. The fermentor is inoculated with a seed culture with a temperature shift from 38 C. to 30 C. for induction. The culture is induced with IPTG one hour after the temperature shift. After the culture reaches an OD600 of 10, the fermentation is fed glycerol at a designated feed rate. When the culture attains an OD600 of 20, the temperature is shifted 30 rpm, and the culture is harvested by continuous centrifugation. OD600 with IPTG for 4 hours. The fermentor is chilled to 15 C., airflow is discontinued and agitation is set to 50 rpm at 15 C., airflow is discontinued and agitation is set to 50 rpm, and the culture is harvested by continuous centrifugation.
(32) Although DNI is expressed in the periplasm of E coli after induction with IPTG, osmotic shock procedures at large scale are problematic and not reproducible. In addition, there is considerable cell lysis during the processing. We have opted to lyse the cells by homogenization and to remove the bulk of the cell debris by continuous centrifugation. The concentrate is then further clarified by TFF using hollow fiber membranes, followed by flat sheet 10 kDa membranes to concentrate the permeate and to diafilter against the first column buffer. The rationale for the HFM is to assist in improving the quality of the DNI collected in the permeate by reducing impurities that would further tax the purification scheme.
(33) The downstream purification process for DNI involves the use of three column chromatography steps, a Mustang filtration step and two UF/DF steps. Capture of DNI is achieved by Q Sepharose FF chromatography. The fractions collected from the Q eluate are pooled based on in-process analysis by SDS-PAGE and passed through a Mustang filter. The Mustang filtered Q eluate pool is then diafiltered into phosphate buffer prior to loading onto a ceramic hydroxyapatite (CHT) column. Fractions collected from the CHT eluate are pooled based on in-process analysis by SDS-PAGE. The CHT eluate pool is adjusted to the appropriate ammonium sulfate concentration by dilution, filtered, and loaded onto the Phenyl Sepharose HP column for final polishing and fractions are collected and pooled based on purity by SDS-PAGE. Final diafiltration into formulation buffer is performed on the Phenyl eluate pool. The formulated bulk is aseptically filtered using a Millipak 0.2 m filter and stored in 1000 ml Nalgene Teflon, PFA Narrow Mouth Bottles at 70 C.
Example V. Formulation of DNI as a Dried Product for Reconstitution
(34) The DNI Drug Product has been formulated using only mannitol and sucrose as excipients and disodium hydrogen phosphate as a buffer. Table 3 lists the quantitative composition of the DNI Drug Product. The bulk drug substance is thawed under 2-8 C. conditions prior to formulation. A 20 mM disodium hydrogen phosphate solution is formulated with disodium hydrogen phosphate and water. The solution is adjusted to a pH of 8.4-8.5. The calculated amounts of mannitol and sucrose are added to the bulk drug substance and mixed until in solution. The solution is adjusted to a pH of 8.1-8.5. Table 4 lists additional elements for inclusion into various emulsion formulation embodiments.
(35) TABLE-US-00003 TABLE 3 Quantitative Composition of the DNI Drug Product Active Ingredients: Quantity: Function: Dominant Negative Inhibitor (DNI) 25 mg Drug Substance Inactive Ingredients: Mannitol, USP 113 mg Excipient Sucrose, USP 33 mg Excipient Disodium hydrogen phosphate (Na2HPO4) 2.4 mg Buffer
(36) TABLE-US-00004 TABLE 4 Animal and Non-Animal Derived Stable Emulsion Formulations SE Formulation Components SE (animal derived sources) SE (non-animal derived sources) Squalene (Shark) Squalene (Olive) Phosphatidylcholine (Egg) Phosphatidylcholine (Soy 95%) Additional formulation excipients (all non-animal derived) Glycerol Glycerol Ammonium Phosphate Ammonium Phosphate Vitamin E Vitamin E Pluronic F-68 Pluronic F-68 Water for Injection Water for Injection
Example VI: Formulation of DNI for Vaccination
(37) The loss of potency of rPA vaccine adsorbed to aluminum adjuvants has been a persistent problem encountered during the advanced development of 3.sup.rd generation anthrax vaccines and is noted in the recent literature by a loss of capacity of rPA vaccine to induce toxin neutralizing antibodies, the key correlate of protective immunity (Wagner, Verma, et al., 2012, Structural and immunological analysis of anthrax recombinant protective antigen adsorbed to aluminum hydroxide adjuvant, Clin Vaccine Immunol, v 19:1465-73). The limitations in recombinant anthrax vaccine development have included rapid loss of potency and effectiveness due to in part to chemical alterations in the rPA protein and subsequent alterations in conformation, limiting the clinical development of purified rPA vaccines. The exact pathways of degradation of rPA vaccine have not been thoroughly characterized, but the large number of asparagine residues has led to the hypothesis that deamidation occurring over time in aqueous buffers can affect protein unfolding and epitope structure and lead to loss of potency and efficacy (Powell, Enama, et al., 2007, Multiple asparagine deamidation of Bacillus anthracis protective antigen causes charge isoforms whose complexity correlates with reduced biological activity, Proteins, v 68:458-79). The anthrax DNI is an analog of rPA containing two mutations that prevent pore formation and translocation of the holotoxin in the cytosol (Sellman, Mourez and Collier, 2001, Dominant-negative mutants of a toxin subunit: an approach to therapy of anthrax, Science, v 292:695-7). DNI binds to the same cell-surface receptors with the same affinity as PA and can form self-assembled heptamers on the surface of cells. The absence of the translocation step prevents DNI-dependent transport of Edema Factor and/or Lethal Factor into cell cytoplasm. Hence, complexes of DNI with LF or EF are nontoxic. DNI was originally proposed for use as an intravenous agent for post-aerosol exposure to anthrax spores to block holotoxin, since one subunit of DNI could block PA-dependent pore formation after heptamerization. It has been shown that animals vaccinated with the DNI antigen induced higher levels of antibodies to toxin and maintained high levels of protective antibody titers for up to one year without booster injections of antigen (Aulinger, Roehrl, et al., 2005, Combining anthrax vaccine and therapy: a dominant-negative inhibitor of anthrax toxin is also a potent and safe immunogen for vaccines, Infection and Immunity, v 73:3408-14). The hyperimmunogencity of DNI has been attributed to the defects in pore formation which may enable more efficient antigen presentation in the context of class II MHC. The anthrax DNI is an analog of rPA containing two mutations that prevent pore formation and translocation of the holotoxin in the cytosol. The DNI protein has been produced at scale as a native protein in E. coli fermentation and a complete battery of release and process control tests has been implemented during its manufacture. In these studies, the effect of synthetic lipid A TLR-4 agonist Glycopyranoside Lipid A (GLA), an analogue of monophosphoryl Lipid A (MPLa), been studied in lyophilized and liquid adsorbed vaccines. The DNI vaccine candidate was formulated in a 9.5 w/v % trehalose 10 mM ammonium acetate buffer pH 7. Aluminum hydroxide adjuvant (Alhydrogel) and aluminum hydroxide with GLA were co-formulated as adjuvants. Lyophilized vaccines were stored at 40 C. for 0, 1, 4, 8, and 16 weeks for immunogenicity studies and also at 70 C. for structural studies. Vaccines were lyophilized to increase their stability at high temperature storage. Liquid vaccines of the same formulations were stored at 40 C. for 0 or 8 weeks.
Example VII: Structural Stability Studies
(38) A study was carried out to test the potential use of lyophilized DNI as a stable vaccine after long-term storage and at extremes of temperature. Physical characterization was carried out for lyophilized and liquid formulations of DNI adsorbed on Alhydrogel and a mixture of Alhydrogel GLA along with a non-adsorbed liquid DNI. The samples were incubated at 4, 40 and 70 C. and sampled at 1, 2, 4, 8, and 16 weeks. The conformational changes in DNI were analyzed by measuring intrinsic tryptophan fluorescence (
Example VIII: Additional Characterization Studies with Lyophilized DNI Vaccine: Particle Size, Glass Transition Temperature, and Adsorption/Desorption Studies
(39) Lyophilized vaccines were characterized for glass transition temperature, protein adsorption, and particle size. The onset glass transition temperature was found to be 115.5 C.1.6 for lyophilized Alhydrogel vaccine formulations and 117.3 C.3.8 for lyophilized Alhydrogel/GLA formulations. Both formulations have high glass transition temperatures similar to pure trehalose, showing minimal water in the lyophilized cakes.
Example IX: Antibody Responses and Temperature Stability of Lyophilized Adsorbed DNI Vaccine
(40) Immunogenicity of the liquid and lyophilized vaccines was tested in Balb/c mice at a single dose level of 10 g per mouse with serological endpoints of total antibodies by anti-PA ELISA (Table 5) and TNA (Table 6). Mice were vaccinated with 10 g DNI dose s.c. on days 0 and 14 and were bled for serum analysis on days 0, 14 and 28. Neutralizing titers (
(41) TABLE-US-00005 TABLE 5 ELISA responses in Balb/c mice immunized (2x) with DNI vaccine adsorbed to AlOH (10 g) DNI DNI- DNI adsorbed DNI adsorbed adsorbed DNI adsorbed unformulated (liquid) (liquid) TLR-4 lyophilized lyophilized TLR-4 1 (day 14) Seroconversion 0/10 3/10 8/9 2/10 8/9 rate (>1:1000) GMT 69 8469 12 4704 Range 1-3,492 176-63,404 1-1,985 1-102,000 2 (day 28) Seroconversion 0/10 4/10 9/9 5/10 10/10 rate (>1:100,000) GMT 1.6 72,448 1,433,709 66,194 1,057,664 Range 1-200 7,112-1,329,414 347,709-7,309,677 9,224-291,047 115,858-15,015,281
(42) TABLE-US-00006 TABLE 6 TNA responses in Balb/c mice immunized (2x) with DNI vaccine DNI DNI- DNI adsorbed DNI adsorbed adsorbed DNI adsorbed unformulated (liquid) (liquid) TLR-4 lyophilized lyophilized TLR-4 2 (day 28) Seroconversion 0/10 8/10 9/9 8/10 10/10 rate (>1:100) GMT 79 503 60 324 Range 1-400 400-1,600 1-400 100-1,600
(43) Newly made liquid (T=0) and lyophilized vaccines produced equivalent immune responses demonstrating that lyophilization does not decrease the immunogenicity of the vaccine. Lyophilized vaccines remained immunogenic even after being stored at 40 C. for 16 weeks. When synthetic MPL is added to the lyophilized formulations during processing, the resulting lyophilized DNI vaccine generated robust immune responses and elicited neutralizing antibodies that are enhanced in relationship to the vaccine that does not contain the synthetic TLR-4 agonist GLA (Tables 3 and 4). The TNA titers after 2 vaccinations with GLA TLR-4 lyophilized as well as liquid vaccine not exposed to temperature stress were 5 to 10-fold higher than in sera from mice vaccinated with the corresponding Alhydrogel vaccines. Furthermore, even when the vaccines were stored at 40 C. for up to 16 weeks prior to administration of the vaccines, there was no evident loss of TNA (
Example X: DNI/GLA-SE Induces High Titer Toxin Neutralizing Antibodies
(44) To evaluate the effect of GLA-SE in conjunction with DNI, Balb/C mice were vaccinated with 5 micrograms of DNI admixed with SE-GLA. SE, or with buffer alone (
Example XI: Combination Vaccine Testing
(45) Groups of ten female Balb/c mice (Jackson Laboratories, Bar Harbor, Me.) with 10 g of either: RiVax, DNI, or a combination of the two, in the presence of the adjuvant Alum (0.85 mg/mL) in PBS. A control group received Alum only (Table 7). Lyophilized dominant negative inhibitor (DNI) was manufactured by Baxter Pharmaceutical Solutions LLC (Bloomington, Ind.). It was stored for 8 years at 4 C. (date of manufacture: Dec. 15, 2003, batch number: 803918A). On the day of use, the lyophilized powder was reconstituted in sterile water. RiVax (lot 190-100L-GLP-FF-090105) and aluminum hydroxide (Alhydrogel) were provided by Soligenix, Inc. (Princeton, N.J.). Alum was provided in histidine buffer (10 mM histidine, 144 mM NaCl, pH 6.0), while RiVax was in 50% glycerol, 50% histidine buffer. All mouse experiments were approved by the Wadsworth Center's Institutional Animal Care and Use Committee (IACUC).
(46) TABLE-US-00007 TABLE 7 Combination Vaccine Testing Results Ricin Endpoint PA Endpoint Ricin Neutralizing LT Neutralizing Survival Group.sup.c Mouse Day 20 Day 200 Day 20 Day 200 Day 20 Day 200 Day 20 Day 200 Ricin LT 1A 1 204800 102400 1 1 1 800 1 1 No 2 204800 204800 1 1 1 1600 1 1 No 3 204800 102400 1 1 1 800 1 1 No 4 204800 51200 1 1 100 100 1 1 No 5 204800 102400 1 1 1 400 1 1 No 1B 1 204800 51200 1 1 1 100 1 1 Yes 2 204800 102400 1 1 1 800 1 1 Yes 3 204800 102400 1 1 1 200 1 1 Yes 4 204800 204800 1 1 1 1600 1 1 Yes 5 204800 204800 1 1 1 400 1 1 Yes 2A 1 1 1 204800 409600 1 1 1600 3200 No.sup.a Yes 2 1 1 204800 204800 1 1 800 800 No.sup.a Yes 3 1 1 204800 204800 1 1 800 1600 No.sup.a Yes 4 1 1 204800 102400 1 1 1600 1600 No.sup.a Yes 5 1 1 204800 102400 1 1 1600 1600 No.sup.a Yes 2B 1 1 1 102400 204800 1 1 200 1600 No 2 1 1 204800 204800 1 1 800 3200 No 3 1 1 204800 102400 1 1 1600 1600 No 4 1 1 204800 204800 1 1 3200 1600 No .sup.5.sup.b 3A 1 51200 102400 102400 51200 1 800 400 800 Yes.sup.a Yes 2 204800 102400 204800 51200 1 800 800 800 Yes.sup.a Yes 3 204800 204800 102400 51200 1 800 100 200 No 4 102400 204800 6400 51200 1 800 200 1600 Yes.sup.a Yes 5 102400 51200 51200 51200 1 400 200 400 Yes.sup.a Yes 3B 1 102400 102400 25600 51200 1 400 1 400 Yes 2 102400 51200 12800 102400 1 100 400 1600 Yes 3 204800 102400 204800 102400 1 400 3200 800 Yes 4 102400 51200 12800 51200 1 200 100 1600 Yes 5 204800 102400 51200 25600 1 400 800 1600 Yes 4A 1 1 1 1 1 1 1 1 1 No 2 1 1 1 1 1 1 1 1 No 3 1 1 1 1 1 1 1 1 No 4 1 1 1 1 1 1 1 1 No 5 1 1 1 1 1 1 1 1 No 4B 1 1 1 1 1 1 1 1 1 No 2 1 1 1 1 1 1 1 1 No 3 1 1 1 1 1 1 1 1 No 4 1 1 1 1 1 1 1 1 No 5 1 1 1 1 1 1 1 1 No .sup.asecond challenge. .sup.bmouse 2B-5 died unexpectedly before blood was collected on day 20. .sup.c1 = RiVax alone, 2 = DNI alone, 3 = RiVax + DNI, 4 = Alum only. Groups labeled with A received lethal toxin in the first challenge, and groups with B received ricin in the first challenge. Titer set to 1 if there was no detectable titer.
(47) Each antigen was mixed with aluminum hydroxide and allowed to adsorb while rotating at 4 C. for 3 hours prior to immunization. Antigens in dual immunizations were adsorbed to Alum separately and mixed before injection. Mice were given a prime immunization, 400 uL i.p., and a boost 2 weeks later. Their immune response was characterized 1 week after the boost, and again 6 months later, by collecting blood from the tail vein.
(48) ELISAs for determining endpoint titers were performed. Plastic plates were coated with either ricin or PA, the blocked with 2% goat serum. Immune serum was then serially diluted two-fold across the plate in duplicate. Secondary antibody detected bound murine-IgG, was visualized by the HRP/TMB calorimetric reaction, and absorbance was detected at 450 nm on a VersaMax spectrophotometer (Molecular Devices, Sunnyvale, Calif.). Endpoint titer was defined as the highest dilution at which the absorbance was still higher than 3 times the background absorbance. On day twenty, six days after boost, anti-ricin titers in the ten mice given RiVax alone were very high, with a geometric mean inverse titer of 204,800. At the same time point, mice given DNI alone also had very high anti-PA titers, with a geometric mean of 187,802. Neither of these groups had any detectable titers against the opposite antigen (Table 8).
(49) TABLE-US-00008 TABLE 8 Geometric Mean Endpoint Titers.sup.a Anti-Ricin Anti-PA Day 20 Day 200 Day 20 Day 200 RiVax 204800 109750 1 1 DNI 1 1 187802 175564 RiVax + 126069 95543 40637 54875 DNI .sup.awhen no titers are detectable, the titer is assigned as 1 for geometric mean calculation
(50) Mice that received the dual immunization with 10 g of each antigen had high titers against both antigens, although the absolute levels were around half of the mice given each antigen by itself. Anti-ricin titers in this group had a geometric mean of 126,069, while anti-PA titers had a geometric mean of 95,543. Therefore, at the endpoint titer level, there is clearly some immune interference from each antigen that impairs the response to the opposite antigen.
(51) Each sample was also tested for neutralizing titers against ricin, in a Vero cell cytotoxicity assay, and lethal toxin, in a J774 cell assay (Tables 9 and 10). Neutralizing titers were defined as the highest dilution in which at least 50% of the cells were protected from the toxin, as defined by the live and toxin killed control wells. Vero cell assays utilized 10 ng/mL of ricin toxin, whereas J774 assays employed 300 ng/mL lethal toxin, with a 1:1 mass ratio of PA and LF. In both assays, 5,000 cells per well were seeded in a 96 well opaque cell culture treated plate in DMEM plus 10% FBS. Immune serum was mixed with toxin at a 1:100 serum dilution, and diluted 2 fold into toxin containing media. In Vero cell assays, the toxin/serum mixture was allowed to incubate with cells at 37 C. for 2 hours, at which point the media was changed. Cell Titer Glo was then used to determine cell viability 48 hours later. In the J774 assay, the media was not changed after toxin/serum mixture addition, and cell viability was determined with Cell Titer Glo 24 hours later.
(52) TABLE-US-00009 TABLE 9 Geometric Mean Neutralizing Titers of Challenge Groups Day 20 - LT.sup.b Day 200 - LT Day 200 - Ricin.sup.c Ricin.sup.a LT.sup.a Total Ricin LT Total Ricin LT Total RiVax 1 1 1 1 1 1 400 528 459 DNI 951 1213 1074 1903 1600 1745 1 1 1 RiVax + DNI 159 264 205 1056 606 800 264 696 429 .sup.atoxin used in particular group; .sup.bLT, Lethal Toxin; .sup.cvery little day 20 ricin neutralizing titers
(53) TABLE-US-00010 TABLE 10 Protection from Toxin Challenges 1st Challenge 2nd Challenge Ricin LT Ricin RiVax 5/5 0/5 N/A DNI 0/4.sup.a 5/5 0/5 RiVax + DNI 5/5 4/5 4/4 Alum Only 0/5 0/5 N/A .sup.aone mouse died unexpectedly before the first tail bleed; N/A, no mice to challenge with that toxin in the second challenge
Example XII. Manufacturing of Lyophilized Stabilized Ricin Vaccine
(54) Colloidal suspensions of aluminum adjuvant particles are unstable and freezing-induced concentration of adjuvant suspensions cause aggregation during freeze-thawing, meaning that conventional lyophilization techniques cannot be successfully applied to vaccines that employ aluminum adjvuants. During lyophilization, aggregation of colloidal aluminum hydroxide suspensions can be inhibited by reducing the extent of their freeze-concentration by using formulations that contain high concentrations of glass-forming excipients, and by limiting the time over which the freeze-concentration occurs by using formulations that contain high concentrations of glass-forming excipients, as well as limiting the time over which the freeze-concentrated suspensions can aggregate by using rapid cooling procedures to maximize the kinetics of glass formation. This process has been developed from examination of a number of parameters and as outlined in
(55) TABLE-US-00011 1. PROCEDURE 1.1. Starting Material Dialysis and Concentration 1.1.1. Prepare Dialysis Buffer (20 mM His mhc, 144 mM NaCl) 1.1.1.1. Dissolve 50.31 g of Histidine monohydrochloride and 101.0 g of NaCl into 12 L of USP water. 1.1.1.2. Adjust the pH of the solution to 6.5 with 1N HCl or NaOH 1.1.1.3. Sterile filter the solution 1.1.2. Slide-A-Lyzer Preparation and loading 1.1.2.1. Hydrate a 70 ml Slide-A-Lyzer (SLD) for two min in the buffer 1.1.2.2. Remove the top of the device and introduce 45 ml of 50% glycerol RiVax stock protein solution 1.1.2.3. Squeeze the membrane lightly to remove any air from the top of the device and close it with the top. 1.1.3. Dialysis 1.1.3.1. Float the SLD in 4 L of sterile HBS 1.1.3.2. Stir the buffer for 2 h at room temperature to allow to equilibrate 1.1.3.3. After 2 h discard the old buffer and replace it with 4 L of fresh dialysis buffer and stir for another 2 h at RT 1.1.3.4. After stirring in fresh buffer for 2 h at RT replace the buffer one last time and move the beaker to 4 C. and allow to stir at 4 C. overnight. 1.1.4. Repeat 4.1.1-4.1.3 in parallel to prepare twice the amount of starting material 1.1.5. Concentration 1.1.5.1. Remove the dialyzed material from the SLD and measure the final volume and concentration of protein (Pierce 23227). 1.1.5.2. Calculate the final volume of concentrate needed to reach a final concentration of 0.5 mg/ml of protein 1.1.5.3. Concentrate the dialyzed material down to the calculated volume with an Amicon ultra centrifugal concentrator 1.1.5.4. Sterile filter the concentrate with a 0.22 um syringe filter to remove any precipitated material 1.1.5.5. Measure the volume and protein concentration of the concentrate (Pierce 23227) 1.2. Prepare solutions 1.2.1. Prepare 50 mL 2X histidine buffered saline (2X HBS, histidine; 40 mM, saline; 288 mM) 1.2.1.1. Add 209.64 mg His mhc to 40 ml of WFI dissolve completely. 1.2.1.2. Add 841.5 mg sodium chloride to the solution and dissolve completely. 1.2.1.3. Adjust the solution to pH 6.5 by adding NaOH or HCl solution dropwise. 1.2.1.4. Bring the final volume to 50 mL by adding WFI. 1.2.1.5. Label the flask 2X histidine buffered saline. 1.2.2. Prepare 2X Antigen Solution in 1X HBS 1.2.2.1. From the concentrate normalize the solution to a 0.4 mg/ml solution using 1X histidine buffer as the diluent 1.2.2.2. Verify the concentration of protein (Pierce 23227) and measure the total volume. 1.2.3. Prepare 200 mL Alhydrogel solution with 3.4 mgAl/ml in 1X HBS 1.2.3.1. Add 68 ml of 10 mgAl/ml stock Alhydrogel to 10 ml of 2X HBS and 3.2 ml of WFI 1.2.4. Prepare 1 L of Histidine Buffer with no sodium chloride (HB) 1.2.4.1. Add 2.094 g of His mhc to 950 ml of WFI and dissolve completely. 1.2.4.2. pH the solution to 6.5 with 1N NaOH 1.2.4.3. Bring the final volume to 1 L by adding WFI 1.2.4.4. Sterile filter the solution 1.2.4.5. Label the flask histidine buffer no saline. 1.2.5. Prepare 200 mL 4X Trehalose in 1X HB. 1.2.5.1. Add 64 g trehalose to 175 ml histidine buffer and dissolve completely. 1.2.5.2. Bring the volume of the final solution to 200 mL by adding 1X HB as needed. 1.2.5.3. Sterile filter the solution 1.2.5.4. Label the flask 4X Trehalose in HB. 1.3. Lyophilizer Preparation 1.3.1. Initialize the cooling system. 1.3.2. Set the condenser to 80 C. 1.3.3. Set the sample shelves to 10 C. 1.3.4. Program the lyophilization parameters found in the automatic program. 1.4. RiVax conjugation 1.4.1. Table 12.4 Ratio of components 4x 2X 4X Alhydrogel RiVax in Trehalose in in 1X HBS 1X HBS IX HB 1 2 1 1.4.2. Mix the Alhydrogel and RiVax Components for this run according to table 12.3.1 1.4.3. Vortex the mixture briefly. 1.4.4. Add a magnetic stir-bar to the flask. 1.4.5. Stir the solutions on the stir plate at 4 C. for 1 h on setting 5. 1.4.6. Assay the mixture for supernatant protein concentration and verify 95% adsorption. 1.5. Washing 1.5.1. Centrifuge the mixture in two 250 ml conical tube on the bench top centrifuge at 3000 rpm for 10 min to pellet the adsorbed adjuvant 1.5.2. Re-suspend the pellet in 450 ml of Histidine Buffer with no salt by pipetting up and down 1.5.3. Centrifuge again at 3000 rpm for 10 min. 1.5.4. Re-suspend the pellet in 2 parts of Histidine buffer(no saline) 1.5.5. After mixing is complete add 1 part 4X Trehalose in 1XHBS. 1.6. Stir for five minutes to mix. 1.7. Aliquot 1 mL conjugated vaccine into 2 ml lyovials and half stopper with slotted stoppers and mark the side of each vial with a permanent marker to distinguish the set 1.8. Aliquot 1 mL WFI into each of 500 lyovials. 1.9. Arrange on the aluminum shelf insert with the dummy vials arranged on the outside of the grouping. 1.10. Insert the aluminum shelf plate with the vial arrangement into the pre-cooled sample shelf. 1.11. Lyophilization 1.11.1. Remove the control program from manual mode and initiate automatic mode with the following drying recipe. Product Name RiVax-TR Product Number RD13-029 Operator Nanotherapeutics R & D Freeze Step 1 2 Shelf Set C. 10 40 Point Ramp Rate C./min 0 0.5 Hold Time Minutes 15 120 Final Freeze C. 40 Setpoint Extra Freeze Minutes 0 Time Starting mTorr 60 Vacuum Setpoint Drying Step 1 2 3 4 5 Shelf Set C. 40 20 20 0 30 Point Ramp Rate C./min 0 0 0 0.2 0.5 Hold Time Minutes 30 30 1200 0 300 Vacuum mTorr 60 60 60 60 60 Setpoint Final Shelf Set C. 24 Point Ramp Rate C./min 0 Vacuum mTorr 60 Setpoint Total Cycle Time 28 h 1.12 Back fill the chamber to 0.5 atm with medical nitrogen. 1.13. Stopper the vials with shelf actuation. 1.14. Break the vacuum directly to the atmosphere. 1.15. Apply an aluminum seal to each of the vials. 1.16. Label each vial with RD13-029 a and b respectively along with the date of manufacture 1.17. Store samples at 80 C.
(56) As used in this specification and in the appended claims, the singular forms include the plural forms. For example the terms a, an, and the include plural references unless the content clearly dictates otherwise. Additionally, the term at least preceding a series of elements is to be understood as referring to every element in the series. The inventions illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms comprising, including, containing, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the future shown and described or any portion thereof, and it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions herein disclosed can be resorted by those skilled in the art, and that such modifications and variations are considered to be within the scope of the inventions disclosed herein. The inventions have been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the scope of the generic disclosure also form part of these inventions. This includes the generic description of each invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised materials specifically resided therein. In addition, where features or aspects of an invention are described in terms of the Markush group, those schooled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. It is also to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of in the art upon reviewing the above description. The scope of the invention should therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. Those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described. Such equivalents are intended to be encompassed by the following claims.