The inactivated dual-antigen API is received by downstream fill-finish sites as a 20–30% v/v packed-cell concentrate of formalin-inactivated Streptococcus equi subsp. zooepidemicus and Streptococcus suis type 2 in phosphate-buffered saline at pH 7.1 ± 0.2. Each 1.0 mL of concentrate typically carries 2.0–5.0 mg total bacterial dry matter, and dilution into injectable suspension is calculated from total protein rather than pre-inactivation CFU because whole-cell clumping after inactivation makes CFU-derived dosing unreliable. For breeding-herd injectables, the antigen concentrate is added to aluminium hydroxide gel (2% Al₂O₃ suspension) to reach a final aluminium concentration of 1.5–2.5 mg/mL, while the antigen payload is adjusted to 250–500 µg total bacterial protein per 2 mL dose. Adsorption is conducted at 4–8°C for 16–24 h with paddle stirring at 80–120 rpm. Adsorption efficiency is confirmed by SDS-PAGE densitometry of the supernatant, with a target of not less than 75% band reduction for both streptococcal antigens. The pH is held at 6.8–7.2 because aluminium hydroxide loses surface charge below pH 6.0 and antigen desorption increases above pH 7.5. The preserved suspension is homogenised through a 100 µm inline filter at 200–400 L/h to remove agglomerates before aseptic filling into 50 mL or 100 mL polyethylene terephthalate bottles.

Sterility is controlled by direct inoculation into thioglycolate medium and soybean-casein digest medium per Ph. Eur. 2.6.1, or by the equivalent USDA 9 CFR Part 113 sterility method, with 14-day incubation at 30–35°C and 20–25°C. The final injectable product must also pass inactivation confirmation: 0.1 mL of bulk is spread onto 5% sheep blood agar and incubated for 72 h at 37°C in 5% CO₂; no viable colonies are permitted. Endotoxin load is monitored by Ph. Eur. 2.6.14, with batch-specific limits fixed in the marketing authorisation because excessive endotoxin from whole-cell Gram-positive bacteria can trigger pyrogenic responses at the injection site. The aluminium hydroxide-adjuvanted format is limited to intramuscular injection in the neck behind the ear; subcutaneous injection is not recommended due to granuloma formation observed at 2–4 weeks after administration. The final product remains shake-test negative after 1 h at 25°C, and sedimentation should not exceed 0.2 mL per 50 mL after 24 h at 25°C.

What Limits Antigen Payload in Oil-Adjuvanted Multivalent Streptococcal Vaccines?

When downstream formulators combine the dual-antigen API with Mycoplasma hyopneumoniae bacterin, Haemophilus parasuis bacterin, or porcine circovirus type 2 capsid protein in a single ready-to-use injectable, the primary constraint is not antigenic identity but the colloidal stability of the oil-adjuvanted emulsion. Water-in-oil formulations use light mineral oil plus mannide monooleate at 8–12% w/w emulsifier in the oil phase. The aqueous antigen phase is added to oil at a 1:1 ratio under high-shear mixing at 10,000–15,000 rpm for 5–10 min at 25°C. Droplet size is measured by laser diffraction; a D50 of 1.0–3.0 µm is maintained for water-in-oil emulsions, and D50 250–500 nm for oil-in-water emulsions. If the aqueous antigen phase exceeds 25% of the final volume or the total protein exceeds 600 µg/mL, the emulsion shows accelerated coalescence and a viscosity rise above 120 cP at 25°C, which can clog 22G vaccine needles during mass vaccination. The dual-antigen API is therefore concentrated to 15–20% packed cell volume before emulsification, and the aqueous phase is prefiltered through 0.8 µm to remove bacterial aggregates larger than the target droplet size.

Compatibility with porcine circovirus type 2 subunit antigen requires pH 7.0–7.4 and an ionic strength below 150 mM NaCl, because high salt destabilises the oil-water interfacial film. Serial mixing order is critical: the antigen phase is blended with phosphate-buffered saline, then added to oil, then passed through a 50 µm screen after emulsification. The final multivalent product is tested for accelerated stability at 37°C for 7 days and 25°C for 6 months. Phase separation greater than 5% v/v at 37°C or a redispersal time greater than 60 s after 24 h at 25°C is considered a batch failure. Container-closure integrity follows Ph. Eur. 3.2.9 for elastomeric closures; a target extractable volume of 2.0 mL per 2.2 mL fill in Type I glass vials is maintained.

Autogenous bacterin manufacturing uses the same dual-antigen API as the starting biological active substance but replaces the master seed with farm-specific isolates of Streptococcus suis type 2 and Streptococcus equi subsp. zooepidemicus that are confirmed by MALDI-TOF and serotype-specific PCR. The API batch is produced from 5–10 L fermenter cultures in Todd-Hewitt broth supplemented with 0.5% yeast extract, harvested at late log phase (OD600 0.8–1.2), and inactivated with 0.3% v/v formaldehyde at 37°C for 24 h. Neutralisation is performed with 0.15% w/v sodium bisulfite, and residual formaldehyde is quantified by a validated acetylacetone method to ensure the final antigen concentrate is below 0.02%. Viability testing requires inoculation of 1.0 mL into 100 mL Todd-Hewitt broth and onto 5% sheep blood agar, followed by 7-day incubation at 37°C and 5% CO₂. Any visible growth in broth or colonies on agar invalidates the batch.

Inactivation kinetic validation is typically performed at three time points: 6 h, 12 h, and 24 h after formaldehyde addition. The table below summarises the control matrix for batch release.

Potency for autogenous injectables is not universally standardised; serological response in target pigs measured by ELISA against capsular polysaccharide is used as a batch release parameter where challenge protection is not ethically feasible. The final autogenous product is usually formulated as a 2 mL intramuscular dose with aluminium hydroxide adjuvant, two doses 3 weeks apart. Mixing with commercial multivalent vaccines in the same syringe is not recommended because antigenic competition and mineral-oil emulsion incompatibility have not been fully characterised for farm-specific isolates. Regulatory acceptance of autogenous bacterins falls under EU Regulation 2019/6 and national provisions for non-immunological veterinary medicinal products; in the United States, autogenous biologics are controlled by applicable USDA Center for Veterinary Biologics requirements.

When Lyophilised API Is Preferred Over Liquid Suspension in Tropical Distribution Chains

Lyophilisation of the inactivated dual-antigen API is selected when downstream fill-finish sites must supply regions with ambient temperatures above 30°C and where cold-chain interruption is likely during final delivery to farms. The liquid antigen concentrate is diluted to 10–15 mg/mL total protein in a cryoprotectant matrix consisting of 6.0% w/v sucrose, 3.0% w/v trehalose dihydrate, 2.0% w/v mannitol, and 1.0% w/v glycine. The bulk is filled at 2.0 mL per 10 mL borosilicate glass vial, frozen on shelf at -45°C for 4 h, and dried with primary shelf temperature at -20°C for 24 h and secondary drying at 25°C for 8 h under 80–100 mTorr chamber pressure. Residual moisture is determined by Karl Fischer titration per Ph. Eur. 2.5.12, with a release limit of less than 3.0% w/w. The dried cake must reconstitute in sterile water for injection within 60 s at 25°C with gentle swirling; incomplete reconstitution or visible particles larger than 50 µm after reconstitution trigger batch rejection.

Antigen recovery after lyophilisation is evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis densitometry and Western blot against both Streptococcus equi subsp. zooepidemicus and Streptococcus suis type 2; a recovery of at least 80% of the pre-lyophilisation band intensity is considered acceptable. Published data for this specific dual-antigen configuration is limited, so downstream users are advised to generate product-specific lyoprotectant screening data before scale-up to 10,000-vial commercial batches. The lyophilised API is reconstituted either with sterile saline for immediate aluminium hydroxide adsorption or with an oil-emulsion diluent for water-in-oil adjuvantation. Vials are stored at 2–8°C in the dry state to maintain a 24-month shelf-life; open vials are discarded after 6 h due to moisture uptake.

Oral Granule and Premix Carriers Require Different Acid-Protection Measures Than Parenteral Formats

Although the primary commercial route for this inactivated dual-antigen API is parenteral, downstream formulation into oral granules, powders, premixes, tablets, and capsules is technically possible for mucosal immune exposure in weaned piglets. The main process challenge is not the antigen itself but the survival of bacterial cell wall integrity through the feed-processing line. Mash mixing at 25–35°C is less destructive than steam-conditioned pelleting at 70–85°C for 15–30 s, which denatures surface proteins and reduces antigenicity. For feed mill integration, the API is therefore applied post-pelleting by spray-drying onto cooled pellets or by mixing into a 1:1000 oral premix with lactose monohydrate and colloidal silicon dioxide. The granulation step uses fluidised-bed top-spray at inlet air 45–50°C and product temperature below 35°C; aqueous binder solution contains 5.0% w/v hydroxypropyl methylcellulose and 0.1% w/v polysorbate 80. The final granules are sieved to 150–710 µm for uniform mixing in creep feed.

Tablet and capsule formats require enteric protection because the inactivated streptococcal cells are partially degraded at gastric pH 2.0–3.5. Direct compression of lyophilised antigen with microcrystalline cellulose, lactose monohydrate, croscarmellose sodium, and 2.0% w/w magnesium stearate yields a core tablet at 8–12 kN compression force; however, compression shear at higher force reduces antigen band intensity by more than 15% in SDS-PAGE. The tablet core is coated with Eudragit L100-55 at 8–10% weight gain using a perforated pan coater at product temperature 28–32°C. Capsule filling with powder is less shear-intensive but requires enteric-coated pellets rather than loose powder to prevent gastric release. Published data for oral booster efficacy of this specific Streptococcus suis type 2 plus Streptococcus equi subsp. zooepidemicus combination is limited, and oral formats are not considered interchangeable with injectable vaccines without target-animal efficacy data filed under EU Regulation 2019/6 or the relevant national registration pathway.

To Maintain Suspension Uniformity in Drinking-Water Delivery Systems

Drinking-water delivery of whole-cell inactivated bacterial antigens presents a physical stability problem that is distinct from feed premixes. The bacterial cells have a mean particle size of 0.8–1.2 µm and settle at approximately 1.5–2.5 mm/h in unthickened water, forming a non-redispersible sediment within 8–12 h. A stock suspension concentrate is prepared by diluting the API to 100–200 µg total protein per 1 L of final drinking water, then adding a stabiliser system of 0.08–0.15% w/v xanthan gum, 0.02% w/v carboxymethylcellulose sodium, and 0.01% w/v polysorbate 80. The final viscosity is maintained below 10 cP at 25°C so that the concentrate remains compatible with proportioner pumps and nipple drinkers. The suspension is mixed with drinking water at a 1:100 ratio, and the diluted product is used within 4–6 h to prevent microbial overgrowth and antigen degradation.

Water quality limits are set at pH 6.5–7.5, free chlorine below 0.5 ppm, total iron below 0.2 ppm, and hardness below 250 mg/L as CaCO₃. Chlorine above 0.5 ppm oxidises surface-exposed streptococcal proteins and can reduce antigen binding in ELISA by more than 20%; iron and hardness promote flocculation of the bacterial cells. Solutions for injection or oral use are prepared fresh from the API concentrate and not stored beyond 24 h at 25°C. As with oral premixes, published efficacy data for drinking-water vaccination against swine streptococcosis with inactivated Streptococcus suis type 2 plus Streptococcus equi subsp. zooepidemicus is limited, and this route should be supported by serological monitoring rather than assumed from injectable challenge data.