VLP Production Platform — R&D Workflow

1. Design the antigen–backbone fusion in silico (linker, dual promoters polh / p10).
2. Synthesise / assemble the fused gene cassettes.
3. Clone the cassettes into transfer Plasmid A (genes 1 & 2) and Plasmid B (genes 3 & 4).
4. Transform E. coli, pick clones, miniprep and sequence-verify (Sanger / NGS).

1. Co-transfect host-cell monolayers in static multi-well plates: Plasmid A + linearised viral DNA in one set of wells, Plasmid B + viral DNA in another (never mixed).
2. Homologous recombination inside the cells packages each plasmid's genes into recombinant Baculovirus Vector A / Vector B.
3. Harvest un-amplified P0 seed per well (~100–200 µL) — separate stocks for Vector A and Vector B.

1. Inoculate host-cell suspension in shaking bottles / T-flasks with P0 seed — a SEPARATE culture for Vector A and for Vector B (kept apart).
2. Maintain continuous agitation (orbital shaking / rocking) to maximise aeration and viability.
3. Perform daily manual or automated sampling to measure cell viability.
4. Harvest at ~60% live/dead ratio. Execute an optional P2 passage if larger volumes are needed.

1. Prepare serial dilutions of Vector A and Vector B working stocks across micro-plates.
2. Inoculate reporter cells and incubate.
3. Measure signal intensity across dilution steps to calculate infectious viral titers (PFU/mL).

1. Co-infect host-cell suspension in large vessels (shake flasks up to 3 L) with Vectors A & B at the calibrated MOI.
2. Incubate under continuous shaking / rocking.
3. Draw daily samples to track host-cell mortality.
4. Terminate culture at the target viability drop — signalling maximum VLP expression and secretion.

1. Centrifuge the bulk harvest to pellet dead insect cells and heavy debris.
2. Collect the VLP-containing supernatant and discard the cell pellet.
3. Process the supernatant through TFF cassettes to concentrate VLPs and remove low-MW contaminants.

1. Run SDS-PAGE and Western blotting to confirm presence and weights of all 4 structural proteins.
2. Prepare negative-stain TEM grids and inspect particle size and spherical structure.
3. Test antigen-binding affinity by SPR or ELISA.
4. Perform safety and endotoxin screening prior to in-vivo mouse immunogenicity studies.

The molecular-biology team designs the antigen fusion and clones it into the two transfer plasmids — this is the ONE step that changes between experiments; everything after it is a fixed pipeline.

Co-transfect the two plasmids SEPARATELY (one set of wells each) to rescue recombinant baculovirus Vectors A and B — kept apart from the very start.

Propagate Vector A and Vector B in TWO separate cultures (never mixed) to build high-titer working stocks — they are only combined later, at Stage 4 co-infection.

Quantify infectious titers of A and B to set an exact 1:1 co-infection ratio — equal delivery of all 4 components.

Co-infect with A & B; ribosomes translate all 4 fused proteins concurrently, which self-assemble into antigen-displaying 3D VLPs.

Separate assembled VLPs from cells and debris — centrifuge out the cell pellet, then concentrate by TFF.

Confirm all 4 proteins are present in ratio and correctly assembled into intact, antigen-displaying particles.

Molecular biologist clones + sequence-verifies Plasmids A & B at the DNA bench.

Operator co-transfects 6-well plates inside the BSC · 2–3 hrs active.

Operator seeds shake bottles into the shaker + daily viability checks.

Operator lays out serial dilutions + reads the fluorometer plate.

Operator co-infects 3 L flasks + samples daily · 3–4 hrs active.

Operator runs the floor centrifuge, then TFF concentration.

Operator runs gels/blots, prepares TEM grids, reads SPR.

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  Thermocycler (PCR)

  Thermal cycler — runs the PCR / assembly reactions and colony-PCR checks during cloning.
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  DNA sequencer

  DNA sequencer — Sanger / NGS confirmation that each plasmid carries the correct fused insert.
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  Multichannel pipette

  Multichannel pipette — parallel liquid handling across plate rows (dilutions, transfers, plating).
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  Conical tubes

  15/50 mL conical tubes — collect and store P0 seed supernatant, media and reagents.

Consumables: Transfer-plasmid backbones A & B · synthetic antigen DNA · assembly / restriction mix · competent E. coli · miniprep + sequencing kits

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  BSC Class II

  Class II biosafety cabinet — HEPA-filtered sterile workspace that protects both the cultures and the operator during open handling.
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  Static incubator

  Static CO₂ incubator — holds sealed culture plates at controlled temperature/CO₂ through the multi-day static rescue.
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  6-well (M6) plates

  6-well (M6) static plates — the vessel for co-transfecting host-cell monolayers at screening scale.
• 
  Conical tubes

  15/50 mL conical tubes — collect and store P0 seed supernatant, media and reagents.
• 
  Multichannel pipette

  Multichannel pipette — parallel liquid handling across plate rows (dilutions, transfers, plating).

Consumables: M6 / M24 static plates · 15/50 mL conical tubes · sterile filter units · pipette tips

• 
  Shaker / rocker incubator

  Orbital shaker / rocker incubator — continuous agitation to aerate suspension cultures and keep cells viable.
• 
  BSC Class II

  Class II biosafety cabinet — HEPA-filtered sterile workspace that protects both the cultures and the operator during open handling.
• 
  Automated cell counter

  Automated cell counter — reads live/dead viability (Trypan Blue) from a microfluidic slide.
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  Microfluidic counting slide

  Microfluidic counting slide — single-use cassette that feeds one sample to the cell counter.
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  Small shaking bottles

  Small shaking bottle — vented vessel for amplifying viral stocks in agitated suspension.

Consumables: Shaking bottles / T-flasks · Microfluidic counting slides · Trypan Blue reagent · Sampling vials · pipette tips

• 
  Microplate fluorometer

  Microplate fluorometer / reader — measures reporter-cell fluorescence to compute infectious titer.
• 
  BSC Class II

  Class II biosafety cabinet — HEPA-filtered sterile workspace that protects both the cultures and the operator during open handling.
• 
  Multichannel pipette

  Multichannel pipette — parallel liquid handling across plate rows (dilutions, transfers, plating).
• 
  Deep-well dilution plates

  Deep-well dilution plate — holds the serial-dilution series for titration.

Consumables: 96-well microplates · deep-well dilution plates · micropipette tips · optical plate seals

• 
  Large shaking incubator

  Orbital shaker / rocker incubator — continuous agitation to aerate suspension cultures and keep cells viable.
• 
  BSC Class II

  Class II biosafety cabinet — HEPA-filtered sterile workspace that protects both the cultures and the operator during open handling.
• 
  Automated cell counter

  Automated cell counter — reads live/dead viability (Trypan Blue) from a microfluidic slide.
• 
  1–3 L shake flasks

  1–3 L shake flask — the largest screening vessel, for the co-infection / VLP-assembly run.

Consumables: 1–3 L shake flasks / bottles · sampling syringes / vials · microfluidic counting slides · Trypan Blue reagent

• 
  High-capacity floor centrifuge

  High-capacity floor centrifuge — pellets dead cells and heavy debris from the bulk harvest.
• 
  TFF filtration skid + cassettes

  TFF filtration skid — tangential-flow cassettes concentrate the VLPs and wash out low-MW contaminants.
• 
  Peristaltic pump

  Peristaltic pump — drives harvest/buffer through the TFF cassettes at a controlled, contamination-free flow.
• 
  Collection bottles

  Collection bottle — sterile vessel for the clarified, concentrated VLP solution.

Consumables: Centrifuge bottles (500 mL–1 L) · TFF cassettes (100–300 kDa MWCO) · sanitary silicone tubing · collection bottles

• 
  Low-voltage TEM (Cryo-EM)

  Low-voltage TEM (Cryo-EM) — direct imaging of particle size and spherical structure (20–200 nm).
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  SDS-PAGE tank

  SDS-PAGE gel tank — separates the 4 structural proteins by molecular weight.
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  Western blot apparatus

  Western blot transfer apparatus — transfers separated proteins to membrane for antibody detection.
• 
  Imaging system

  Gel / blot imaging system — captures chemiluminescent band images for QC records.
• 
  SPR / ELISA reader

  SPR / ELISA reader — measures the antigen-binding affinity of the displayed surface antigens.

Consumables: Polyacrylamide gels · PVDF membranes · carbon-coated TEM grids · chemiluminescent substrates · micro-tubes · filter paper