β-Propiolactone (BPL) Viral Inactivation — Standard Operating Procedure (SOP)

1. Purpose

Viral inactivation refers to physical or chemical treatments that render viruses non-infectious. It is commonly used to prepare viral antigens and to detoxify virus-contaminated cells or tissues. Typical approaches include:

• Formaldehyde (paraformaldehyde) inactivation: crosslinks proteins to disrupt viral structures.
• β-Propiolactone (BPL) inactivation: alkylates and damages nucleic acids, inhibiting replication.
• Ultraviolet (UV) inactivation: induces nucleic-acid lesions that block viral proliferation.

These methods can be used alone or in combination to maximize safety while preserving antigenicity as much as possible. This SOP specifies the principles and management requirements for viral inactivation using β-propiolactone (BPL).

2. Scope

• Applicable to chemical inactivation of a wide range of DNA/RNA, enveloped/non-enveloped viruses.
• Applicable to virus-containing matrices such as cell-culture supernatants, tissue homogenates, serum/protein solutions, and vaccine bulks.
• Applicable to preparation of inactivated antigens (e.g., vaccine or diagnostic antigens) and to deactivation of materials contaminated by viruses.
• Suitable where preservation of protein epitopes is important (subject to project-specific validation).
• Suitable for process flows that can accommodate BPL hydrolysis/residual-removal steps.

3. Biosafety and Responsibilities

• Biosafety level:H5/H7 highly pathogenic avian influenza viruses and H2N2 subtype: perform in BSL-3 laboratories.Other influenza viruses: perform in BSL-2 laboratories.
• Personal protective equipment (PPE): compliant lab gown, disposable cap/shoe covers, double latex/nitrile gloves, N95 or equivalent respiratory protection, and safety goggles/face shield.
• Chemical safety (BPL): BPL is a strong mutagen and a suspected carcinogen; it is volatile and highly reactive. All preparation and additions must be performed in a fume hood with an ice bath. Avoid skin/mucosal contact and inhalation. Prepare a spill kit (absorbent materials, alkaline neutralizer, sealable waste containers).

4. Materials and Equipment

4.1 Reagents and Consumables

• 9–10-day-old SPF chicken embryos
• β-Propiolactone (BPL): prepare fresh 2% (w/v) solution (2 g BPL in 100 mL double-distilled water pre-chilled on ice). Keep on ice and work in a fume hood throughout.
• 0.5 M disodium hydrogen phosphate (Na₂HPO₄)
• 7% NaHCO₃ solution (for pH adjustment)
• Sterile PBS (pH 7.2–7.4)
• 75% ethanol
• T75 cell-culture flasks (for subsequent safety testing/culture)
• Red blood cells and HA plates (for hemagglutination assay)
• Sterile centrifuge tubes, pipettes/sterile tips, labels, etc.

4.2 Instruments

Candling lamp, egg opener, balance, ultracentrifuge/bench centrifuge, 37 °C water bath (with 15-min mixing reminders), 4 °C refrigerator and ice-bath setup, biological safety cabinet, fume hood, pH meter.

5. Procedure (BPL Inactivation)

General: Perform all open manipulations in a biological safety cabinet. Prepare and add BPL only in a fume hood with an ice bath.

（1）Virus preparation: Obtain clarified viral fluid. Record batch number, titer, volume, and source embryo information.

（2）Pre-inactivation setup: Pre-chill double-distilled water and ice bath. In a fume hood, prepare 2% BPL (2 g/100 mL ice-cold ddH₂O), mix, and keep on ice. Prepare 0.5 M Na₂HPO₄ and 7% NaHCO₃. Set the 37 °C water bath and set a 15-min mixing reminder.

（3）Dosing and addition: Transfer 38 parts of virus fluid to a labeled sterile container. Add 1 part 0.5 M Na₂HPO₄ and mix gently. In the fume hood on ice, add 1 part of 2% BPL (final BPL concentration 0.05%). Mix immediately and thoroughly to ensure contact with viral particles. Record all volumes and addition times.

（4）Inactivation reaction (choose one; do not combine):

• Option A: Incubate at 37 °C for 2 h, gently invert several times every 15 min.
• Option B: Incubate overnight (≥12 h) at 4 °C without agitation.

Note:Keep containers closed to prevent volatilization and exposure.

（5）pH adjustment: After reaction, in the biosafety cabinet slowly titrate with 7% NaHCO₃ to adjust the mixture to pH 7.3–7.4 (verify with a calibrated pH meter). Mix gently.

（6）Verification of inactivation: Blind-pass in chicken embryos for three generations; proceed only when hemagglutination (HA) testing is negative at every passage, at which point declare “inactivation complete” and allow progression to subsequent steps/release.