| HS Code | 375179 |
| Product Name | Anthrax Spore Vaccine, Live (Noncapsulated Strain) Veterinary Grade API |
| Product Category | Veterinary biological active pharmaceutical ingredient (API) |
| Active Ingredient | Live spores of a noncapsulated strain of Bacillus anthracis |
| Strain Type | Noncapsulated (capsule-deficient) attenuated anthrax strain |
| Immunogenicity | Induces protective immunity against virulent encapsulated anthrax strains |
| Target Species | Livestock and veterinary animals such as cattle, sheep, goats, and horses |
| Veterinary Grade | Suitable for veterinary pharmaceutical formulation use |
| Dosage Forms | Tablets, injections, capsules, powders, granules, premix, and solutions |
| Shelf Life | Typically 12–36 months depending on final formulation and stability data |
As an accredited Anthrax Spore Vaccine,Live(Noncapsulated Strain) Veterinary Grade API for Tablets / Injections / Capsules / Powders / Granules / Premix / Solutions factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | Supplied in 1 kg, 5 kg, or 25 kg airtight, tamper-evident containers; sterile vials for injections; labelled for veterinary use. |
| Container Loading (20′ FCL) | 20′ FCL loading: refrigerated at 2–8°C, palletized and secured; segregated from food/feed, with biohazard labels and complete shipping documentation. |
| Shipping | Anthrax spore vaccine, live (noncapsulated strain), veterinary-grade API — bulk active material for production of tablets, injections, capsules, powders, granules, premix, or solutions. Transport at +2°C to +8°C in leak-proof triple packaging labelled UN 3373 ‘Biological Substance, Category B.’ Follow applicable live-biological shipping, export/import, and veterinary/biosecurity requirements. |
| Storage | Store under refrigerated conditions at 2–8°C, protected from light and moisture. Do not freeze or expose to excessive heat. Keep in tightly sealed, original containers away from incompatible substances. Ensure cold chain integrity during transport and handling. For veterinary use only. |
| Shelf Life | Shelf life is typically 24 months from manufacture when stored at 2–8°C, protected from light, and kept unopened. |
During aseptic formulation of the noncapsulated live spore API into a parenteral suspension, the finished product is compounded as a low-shear blend of washed spore concentrate and a stabilised diluent in a jacketed 316L stainless steel vessel equipped with a bottom-mounted magnetic impeller operating between 50 rpm and 150 rpm; higher shear rates generate cavitation that damages the spore coat. The aqueous matrix typically includes saponin at 0.5%–2.0% w/v as the adjuvant, sodium chloride at 0.3%–0.9% w/v to achieve an osmolality of 280–320 mOsm/kg, and glycerol at 10%–20% v/v where freeze-thaw protection is required during cold-chain transport. The spore concentration is adjusted after plate enumeration to meet the minimum dose specified in 9 CFR 113.65, which for the Sterne 34F2 noncapsulated strain is not less than 1 × 10^6 CFU per dose in the final container. Sterility testing is performed by membrane filtration according to Ph. Eur. 2.6.1 and USP <71> with incubation at 30–35°C for 14 days; the vaccine is not terminally sterilised. Homogeneity is monitored by aseptic sampling from the top, middle, and bottom dip ports of the compounding vessel, and the acceptance criterion is a coefficient of variation of less than 10% in viable spore count across the three sample ports. During filling, the suspension is maintained under continuous agitation and transferred through a peristaltic pump equipped with platinum-cured silicone tubing into Type I borosilicate vials of 10 mL, 20 mL, or 50 mL nominal volume, sealed with chlorobutyl rubber stoppers in a Grade A laminar-airflow zone. Filled vials are held at 2–8°C; forced degradation studies under VICH GL17 indicate that a temperature excursion to 25°C for 7 days produces a potency loss exceeding 0.5 log10, although the exact slope varies with residual moisture and saponin load.
Lyophilisation of the noncapsulated spore API into a powder for reconstitution is governed primarily by the glass transition temperature of the protective matrix rather than by spore thermal resistance alone. Sucrose at 5%–10% w/v, trehalose at 5% w/v, and monosodium glutamate at 1%–2% w/v are added to the bulk spore suspension before filling because they depress the melting point of the frozen matrix and maintain an amorphous phase during drying. The suspension is filled in 3 mL or 5 mL volumes into 10R glass vials and loaded onto stainless steel shelves pre-cooled to 5°C. Freezing proceeds at a shelf ramp of 0.5°C/min to -45°C and is held for 180 min; annealing at -20°C for 60 min is sometimes inserted to allow ice crystal growth and reduce primary drying resistance. Primary drying is conducted at a shelf temperature between -15°C and -20°C with a chamber pressure of 50–100 µbar for 20–36 h; the endpoint is determined by comparative pressure measurement with Pirani and capacitance manometer sensors exhibiting a ΔP of less than 5 µbar. Secondary drying at 25°C for 6 h reduces residual moisture to 1.0%–2.5% as measured by Karl Fischer titration according to USP <921>. In production-scale lyophilisers with shelf area of 2–5 m², batch-to-batch variation in primary drying time of ±3 h is observed when condenser coil ice loading exceeds 80% capacity; shelf temperature uniformity must remain within ±1°C under defined IQ/OQ protocols. The dried cake is required to reconstitute in sterile water for injection in <60 s with no visible aggregates. Viable spore recovery after lyophilisation and reconstitution typically exceeds 70% of the pre-dry count when the protective matrix remains amorphous with a glass transition onset above 40°C; crystallisation of sucrose leads to cake collapse and spore inactivation beyond 1 log10.
| Quality attribute | Injectable suspension | Lyophilised powder | Unlicensed oral premix |
|---|---|---|---|
| Viable spore count | Not less than 1 × 10^6 CFU per 1 mL dose per 9 CFR 113.65 | Not less than 1 × 10^6 CFU per reconstituted dose | No monograph specification; feasibility data only |
| Residual moisture | Not applicable (liquid) | 1.0%–2.5% w/w by Karl Fischer (USP <921>) | <3.0% w/w for granule stability |
| Storage temperature | 2–8°C | 2–8°C | 2–8°C for experimental baits |
| Sterility / bioburden | Sterile per Ph. Eur. 2.6.1 | Sterile per Ph. Eur. 2.6.1 | Low bioburden only; no sterility claim |
Dry powder handling of the lyophilised or spray-dried spore API prior to filling into sachets, capsules, or bulk containers introduces mechanical stress from milling, sieving, and blending that reduces viable spore count through shear and desiccation. Particle size distribution is controlled between 50 µm and 150 µm by low-energy conical milling at 300–500 rpm; spore viability retention across this step is measured by pour plate enumeration on polymyxin–lysozyme–EDTA–thallous acetate agar at 37°C for 24–48 h. Powder flowability for capsule or granule filling is evaluated by Hausner ratio and Carr index; a Hausner ratio above 1.25 triggers addition of 0.5%–1.0% w/w colloidal silicon dioxide, which has not shown incompatibility with spore viability in short-duration blending trials at 20–25°C. Granulation of the spore API with microcrystalline cellulose and lactose monohydrate using a top-spray fluidised bed at inlet air temperature 35°C and product temperature 25–30°C must avoid wet mass moisture above 10% w/w because excess free water promotes premature germination of spores and subsequent inactivation during drying. The resulting granules are filled into hard gelatin capsules or aluminium sachets under controlled relative humidity ≤40% at 20°C. Loss on drying is maintained at <3.0%; oxygen headspace in sealed sachets is reduced to <1% by nitrogen flushing to limit oxidative damage to the spore coat. Published stability data for granulated oral dosage forms of this specific noncapsulated anthrax spore vaccine are limited; the parameters described are derived from standard lyophilised veterinary vaccine powder handling rather than a licensed oral granule product.
Because oral administration of a live spore anthrax vaccine must survive gastric pH 2.0–3.5 and bile salt concentrations of 5–10 mM in the duodenum, the noncapsulated Sterne 34F2 spore API is not authorised in most jurisdictions as a feed premix or oral drench under USDA 9 CFR 113.65 or Ph. Eur. 0441; published data for enteric-coated capsule or lipid-encapsulated spore delivery in ruminants are limited to experimental challenge studies without commercial license. Where a solution or drench is prepared extemporaneously by reconstituting lyophilised powder in tap water or saline, spore sedimentation in 1–4 h and loss of viability at ambient temperatures above 25°C impose a use window of ≤2 h. No commercial oral premix specification currently exists; formulations claiming extended spore survival in feed matrices would require demonstration of viability under VICH GL17 stability protocols before regulatory acceptance.
Evaluation of tablet compression for live spore API delivery is restricted to research formulations because conventional direct compression at 10–25 kN compaction force reduces spore viability by 1–3 log10 due to mechanical shear and local temperature rise. Where tablets are considered for oral veterinary administration, the noncapsulated spore API must first be adsorbed onto a porous carrier such as microcrystalline cellulose with a specific surface area of 1.1–1.3 m²/g and then blended with croscarmellose sodium at 2%–5% w/w as disintegrant before compression. The compression mixture is processed on a rotary tablet press with turret speed 10–20 rpm and pre-compression force 2–5 kN to form tablets of hardness 30–50 N and friability <1.0% per USP <1216>. Tablet thickness is maintained at 4.0–5.0 mm; dissolution testing in 0.1 N HCl for 60 min shows that unprotected spores are inactivated within 15 min, whereas enteric coating with Eudragit L 30 D-55 applied to 8%–10% weight gain delays release until pH >5.5. Capsule filling of spore-containing granules into size 0 or 00 hard gelatin capsules is performed on a dosator-type capsule machine at 2,000–5,000 capsules/h; the main quality risk is moisture ingress through the gelatin shell when stored above 60% RH, which activates spore germination and reduces shelf life. Because no licensed tablet or capsule product exists for anthrax spore vaccine in veterinary medicine, these processing parameters are derived from published feasibility studies on live biotherapeutic spores and are not codified in 9 CFR 113.65.
Routinely, cold-chain distribution of finished dosage forms containing the Sterne 34F2 noncapsulated spore API is executed in insulated expanded polystyrene shippers with validated phase-change materials maintaining an internal temperature envelope of 2–8°C for 48–96 h; temperature excursions above 8°C are recorded by USB data loggers with ±0.5°C accuracy calibrated to ISO/IEC 17025. Injectable suspensions are shipped in secondary packaging with 10 mm foam inserts to limit mechanical vibration, because continuous agitation at frequencies above 5 Hz can resuspend settled spores but also increase shear-induced loss of viability when air bubbles are entrained. Lyophilised vials are less sensitive to vibration but require protection from moisture ingress; desiccant sachets containing silica gel with a moisture capacity of ≥20% w/w are placed in each aluminium pouch. The cold-chain stability data generated under VICH GL17 for lyophilised powder demonstrate acceptable potency for 24 months at 2–8°C, while liquid injectable suspensions typically have a shelf life of 12–18 months at the same condition. For intermediate API powder stored in bulk, split shipments in 1 kg or 5 kg laminated foil bags with nitrogen headspace <1% oxygen prevent cross-contamination and reduce the risk of temperature cycling condensation. No terminal sterilisation step is applied to the finished dosage form because autoclaving at 121°C for 15 min inactivates spores; therefore, all downstream handling from API release through final packaging must maintain aseptic or low-bioburden conditions.
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Anthrax Spore Vaccine, Live (Noncapsulated Strain) Veterinary Grade API is a lyophilized spore concentrate prepared from Bacillus anthracis Sterne 34F2, a pXO2-deficient seed lot that does not synthesize the poly-γ-D-glutamic acid capsule but retains the pXO1 toxin operon. The bulk material is released for further veterinary formulation into injections, reconstitutable powders, solutions, premixes, granules, capsules, and tablets. The noncapsulated phenotype is verified on bicarbonate agar under 5% CO₂; capsule-negative colonies are selected as the working seed. Sporulation is performed on a defined sporulation medium at 37 °C until phase-contrast microscopy indicates ≥90% free refractile spores. The harvested biomass is washed with sterile water, concentrated by tangential-flow filtration, and lyophilized with a protective matrix consisting of sucrose and monosodium glutamate. The resulting API is a free-flowing powder with a spore count that must be correlated to protective potency. Residual moisture after lyophilization is controlled by Karl Fischer titration, and the material is stored at 2–8 °C under desiccant.
The API model designation follows the Sterne 34F2 seed lot and is supplied as a concentrated spore powder with a suitability certificate for veterinary formulation. The material is not intended for direct administration without downstream dilution or formulation. Final product forms include subcutaneous injectable suspensions prepared from reconstituted lyophilized powder, oral powders and granules for drench or feed top-dressing, and experimental solid oral capsules or tablets. Injections and solutions require sterile diluent and are prepared immediately before use because extended holding of reconstituted spores at room temperature reduces viability. Powders, granules, and premixes are intended for homogeneous incorporation into feed or for individual oral dosing. The uniformity of spore distribution must be verified by assay of multiple blend samples according to the registration file. Lyophilized API is typically assigned a shelf life established by real-time stability; accelerated stability studies at 25 °C/60% RH are used to support excursions. The final dosage form retains potency only if the container closure system limits moisture ingress below 3.0% w/w.
Release of this veterinary API is governed by the registration file, which commonly references European Pharmacopoeia monograph 0441 for anthrax spore vaccine (live) for veterinary use, the OIE Terrestrial Manual Chapter 3.1.1, and where applicable USDA 9 CFR 113.65. Identity is confirmed by gamma bacteriophage lysis on sheep blood agar and by absence of capsule production on bicarbonate agar after 18–24 h at 37 °C in 5% CO₂. Purity assessment uses sterility testing for parenteral presentations or microbial enumeration for non-sterile oral and feed presentations according to Ph. Eur. 2.6.1, Ph. Eur. 2.6.13, or corresponding VICH harmonized methods. The spore count is enumerated after heat shock at 65 °C for 15 min to destroy vegetative cells; dilutions are plated on PLET agar and incubated at 37 °C for 48 h. The minimum count is batch-specific and validated against the potency test; commonly formulated doses are not less than 1 × 10⁷ CFU per dose when reconstituted, although some monographs allow a wider range based on challenge data. Potency is demonstrated by active protection in immunized guinea pigs or mice against a virulent B. anthracis challenge, with survival at 14 days post-challenge compared with unvaccinated controls. Safety testing includes injection of a defined excessive dose into guinea pigs and observation for 7 days for local or systemic adverse reactions; capsulated revertants or heavy extraneous contamination cause batch rejection.
| Parameter | Analytical Control |
|---|---|
| Identity | Gamma phage lysis on sheep blood agar; capsule-negative growth on bicarbonate agar under 5% CO₂ |
| Viable spore count | Heat shock 65 °C for 15 min; PLET agar; 37 °C for 48 h; count correlated to potency |
| Residual moisture | Karl Fischer titration; release limit ≤ 3.0% w/w |
| pH after reconstitution | 6.0–7.5 |
| Potency | Mouse or guinea pig active challenge; survival fraction or PD₅₀ versus reference vaccine |
| Safety | Overdose safety in guinea pigs; 7-day observation for adverse reactions |
| Purity | Ph. Eur. 2.6.1 sterility or Ph. Eur. 2.6.13 microbial enumeration; absence of capsulated revertants |
Formulation of a live spore API into liquid or solid veterinary dosage forms is constrained by the spore’s susceptibility to heat, shear, and prolonged high water activity. In aqueous solution or suspension, the spores remain viable only if the formulation pH is maintained between 6.0 and 7.5 and the product is held at 2–8 °C; preservatives such as benzyl alcohol, parabens, and phenolic compounds can reduce viability and must be excluded or demonstrated compatible by spore count recovery over the intended shelf life. For injectable presentations, the final dose is typically prepared as a subcutaneous suspension in sterile diluent. Intravenous or intramuscular administration is not indicated for this live spore product; the target tissue is the subcutaneous compartment, where local phagocytosis of noncapsulated spores initiates protective immunity. Liquid formulations manufactured aseptically cannot be terminally sterilized by filtration because the spore size exceeds the pore diameter of sterilizing-grade membranes; aseptic processing is therefore required.
Dry processing of the API into granules, powders, or premixes requires low-water-activity excipients with water activity ≤0.3, low-shear blending, and avoidance of temperatures above 40 °C for extended periods. Wet granulation with aqueous binders is generally unsuitable because germination and vegetative growth may occur; if granulation is necessary, fluid-bed drying with inlet air below 35 °C and rapid moisture removal is used. For feed premixes, the carrier should be selected to avoid free water and antimicrobial additives. Ionophore coccidiostats and antibiotic growth promoters may be incompatible and should not be combined with the live spore API unless compatibility data exist. Blend uniformity is assessed by sampling 10 locations and performing heat-shock spore count on PLET agar. Each sample should recover the target spore count within the acceptance range defined by the batch release specification.
Tablet and capsule presentations containing live B. anthracis spores are atypical for veterinary immunoprophylaxis; the established commercial format is a lyophilized plug or liquid suspension for subcutaneous injection. Where a solid oral dosage form is required, the formulation must be designed around survival during compaction. Direct compression on a rotary tablet press is conducted at the lowest compression force that produces acceptable friability. Published data for this specific configuration is limited, so pre-compression spore recovery screening is required before any compression force is fixed. Instrumented press studies should measure ejection force, die-wall residual pressure, and tablet hardness; spore viability is then assayed by heat-shock plate count on PLET agar after compression. Excipients such as mannitol and low-moisture microcrystalline cellulose are preferred over high-moisture binders. Magnesium stearate is used at the minimum effective level, commonly 0.25–1.0% w/w, because prolonged mixing with hydrophobic lubricants can reduce dispersibility and delay spore release in dissolution media.
Encapsulation into hard gelatin or HPMC capsules provides lower shear than tableting and is more compatible with live spore viability; fill weight is adjusted to deliver the target spore count per capsule with an overage to account for the validated loss during filling. Film-coating of tablets is generally avoided because pan temperature and aqueous spray can reduce viability; if enteric protection is required, dry enteric coating technologies or capsule-in-capsule approaches are evaluated. Lyophilized API can be dry-mixed with microcrystalline cellulose and mannitol, but the mixture must be stored at 2–8 °C in moisture-barrier packaging. Moisture ingress above 3.0% w/w accelerates spore germination and viability loss. Roller compaction is preferred over wet granulation for solid oral forms; the compaction pressure is set during formulation development by measuring post-compaction spore recovery, because published B. anthracis-specific thresholds for roller compaction pressure and tablet compression pressure are limited.
For oral powders and granules, the API is typically blended with a non-hygroscopic carrier such as anhydrous lactose or calcium carbonate. The final product should be packed in unit-dose sachets or foil-lined pouches to limit moisture and oxygen exposure. In feed premixes, the spore-containing fraction is diluted into a carrier that does not generate heat during mixing. Mixing time is optimized by blend uniformity studies rather than fixed by generic mixing rules. If the premix is to be pelleted, the pellet mill conditioning temperature must be controlled below 40 °C and the dwell time minimized, because moist heat at conditions above this range reduces viable spore count. Post-pelleting spore recovery is part of process validation.
The principal difference between this API and other anthrax veterinary biologics is the absence of the pXO2-encoded poly-γ-D-glutamic acid capsule in the Sterne 34F2 seed. Capsulated field isolates evade phagocytosis and produce progressive infection; the noncapsulated vaccine strain is susceptible to macrophage clearance in immunocompetent animals but retains the pXO1 toxin genes, allowing transient synthesis of protective antigen, lethal factor, and edema factor during local replication. This distinguishes the live spore product from inactivated anthrax vaccines or purified protective antigen preparations, which primarily present a single antigen and require adjuvants to achieve acceptable immunogenicity. Live spore vaccination generally induces a broad response against toxin components and spore-associated antigens, but it is contraindicated in animals receiving antimicrobial therapy because the vaccine strain is susceptible to β-lactams, tetracyclines, and fluoroquinolones. Unlike capsulated Pasteur-derived vaccines, the noncapsulated product has a lower risk of progressive disease in vaccinated animals, although accidental self-injection in humans still requires medical evaluation because of the toxigenic potential.
Compared with anthrax vaccine adsorbed for human use, which is an aluminum-adjuvanted filtrate containing protective antigen, this veterinary live spore API presents a replicating immunogen and is not interchangeable. The veterinary live product cannot be used in human medicine. Differences from capsulated strains and purified antigen products also extend to batch-release potency models: live spore vaccines are controlled by active challenge, whereas purified antigen products are controlled by in vitro antigen content and adjuvant quality. The noncapsulated phenotype is a critical safety marker; any appearance of mucoid, capsulated colonies during seed lot testing or final product testing indicates reversion or contamination and is a rejection criterion. The API must be handled under biosafety level 2 containment because the live spores remain toxigenic and can cause laboratory-acquired infection if mishandled. All equipment surfaces, filters, and waste streams should be decontaminated with sporicidal disinfectants validated against B. anthracis spores.
