I. Introduction

The baculovirus expression system (BVES) has emerged as a core tool for the production of recombinant proteins, recombinant adeno-associated virus (rAAV) vectors, and subunit vaccines (e.g., the influenza vaccine FluBlok®), thanks to its high expression efficiency, excellent protein modification capabilities, and biosafety. In the application of BVES, baculovirus titer assay is an irreplaceable quality control step—baculovirus titer (the number of infectious viral particles per unit volume) directly determines the infection efficiency of insect host cells, the yield of target proteins, and the stability of production batches. During the baculovirus infection cycle, budded virions (BVs) are the core form mediating exogenous gene expression in vitro, and the accurate determination of their activity and concentration is a key prerequisite for ensuring the efficiency and safety of biopharmaceutical production.

II. Technical Background of Baculovirus Titer Assay

(I) Necessity of Titer Assay

The infection efficiency of baculoviruses (such as the commonly used Autographa californica nuclear polyhedrosis virus, AcNPV) entirely depends on the activity and concentration of BVs. Titer assays are required to control key nodes throughout the entire production process:

Recombinant Virus Screening: Verify the activity of purified recombinant viruses to eliminate defective viruses;

Virus Amplification: Monitor the proliferation efficiency of BVs to ensure the titer reaches 10⁶–10⁸ PFU/mL, meeting the needs of large-scale infection;

Cell Infection: Calculate the multiplicity of infection (MOI) based on titer—an excessively high MOI causes premature cell lysis, while an excessively low MOI reduces protein expression efficiency. Precise control of this ratio is critical for process stability.

(II) Correlation Between Host Cells and Assay

Titer assays rely on sensitive insect cell lines, and cell characteristics directly affect assay accuracy:

Sf9 Cells: Highly sensitive to AcNPV, with regular morphology during adherent growth, making them the first choice for virus amplification and titer assay;

Sf21 Cells: Larger in diameter than Sf9 cells, with more observable plaques, suitable for refined detection;

Hi5 Cells: More suitable for the expression of secretory proteins, but less sensitive to baculoviruses, so they are rarely used in titer assays.

III. Common Methods for Baculovirus Titer Assay

(I) Plaque Assay: The "Gold Standard"

Based on the principle that "a single infectious BV forms a visible plaque after infecting cells," results are expressed as PFU/mL. The procedure is as follows:

Seed logarithmic-phase Sf9 cells (2×10⁵ cells/mL) into plates and incubate for 4–6 hours to form a monolayer;

Perform 10-fold serial dilution of the virus solution (10⁻²–10⁻⁸), with 3 replicate wells per dilution. Add 100–200 μL of the diluted virus solution to each well and incubate at 37°C for 1 hour;

Aspirate the virus solution, add medium containing low-melting-point agarose (at 40°C), and incubate at 27°C for 5–7 days after solidification;

Count the dilution with 10–100 plaques and calculate the titer using the formula: "Titer = Number of plaques × Dilution factor / Infection volume."

Its advantage lies in directly quantifying infectious viruses with reliable results; however, it is time-consuming (5–7 days), relies on manual counting, and has poor adaptability to high-throughput testing.

(II) End-Point Dilution Assay: A Semi-Quantitative Alternative

Titer is inferred by detecting the minimum dilution that causes cytopathic effect (CPE), with results expressed as TCID₅₀/mL:

Seed Sf21 cells into 96-well plates (1×10⁴ cells/well) and incubate for 24 hours to allow adherence;

Perform 2-fold or 10-fold serial dilution of the virus solution (10⁻¹–10⁻¹²), with 8–12 replicate wells per dilution. Add 100 μL of the diluted virus solution to each well;

Incubate at 27°C for 7–10 days and record the number of wells with CPE;

Calculate TCID₅₀ using the Reed-Muench method and convert it using the relationship "1 TCID₅₀ ≈ 0.7 PFU."

Its advantage is that it does not require agarose overlay and is easy to operate; however, it has a long cycle (7–10 days), is semi-quantitative, and has slightly lower accuracy.

(III) Rapid Detection Technologies: Efficiency-Upgraded Solutions

Immunofluorescence Assay (IFA): At 24–48 hours post-infection, use antibodies against viral structural proteins (e.g., anti-Polh antibodies) combined with fluorescent secondary antibodies to count positive cells and infer titer. The cycle is shortened to 2–3 days with high sensitivity, but it requires specific antibodies and has high costs;

Quantitative Real-Time PCR (qPCR): Detect the copy number of conserved viral genes (e.g., Polh, p10) and obtain the infectious titer using a conversion factor (1×10⁶ copies ≈ 10³–10⁴ PFU). It can be completed within 6 hours with high throughput, but it cannot distinguish between infectious and non-infectious viruses and needs verification with traditional methods.

IV. Application of Titer Assay in Biopharmaceutical Quality Control

(I) Production Process Control

rAAV Vector Production: The BV titer needs to reach above 10⁷ PFU/mL to achieve efficient packaging in Sf9 cells (yield: 10¹⁴ VG/L);

Subunit Vaccine Production: For example, in the production of FluBlok®, MOI (5–10) is controlled based on titer to ensure efficient expression of hemagglutinin (HA) protein in Sf9 cells;

Recombinant Protein Production: Adjust the infection time according to titer to avoid premature cell lysis affecting protein secretion.

(II) Host Residue Risk Management

The Chinese Pharmacopoeia (2020 Edition) stipulates that DNA residues in biological products should be ≤ 100 pg/dose, and the FDA and EP also have strict limits. If the virus titer is too low, infection efficiency decreases and cell culture is prolonged, increasing the release of residual host cell DNA. Accurate titer assay can optimize the infection process, indirectly reduce residual risks, and ensure product safety.

V. Summary and Outlook

Baculovirus titer assay serves as the "activity benchmark" for BVES production. The evolution from traditional plaque assays to rapid molecular detection methods not only retains accuracy but also meets the needs of efficient production. In the future, with the expanded application of BVES in gene therapy and novel vaccines, titer assays will move toward automation (e.g., microfluidic single-virus counting) and standardization (multi-center data calibration), providing more precise quality control support for the efficient R&D and large-scale production of biopharmaceuticals.

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