Baculovirus BIIC Banking

Titerless Infected-Cells Preservation (TIPS) / Baculovirus-Infected Insect Cell (BIIC)

Advantages of Using TIPS/ BIIC

The titerless infected-cells preservation and scale-up (TIPS) also known as Baculovirus-infected insect cells (BIIC) (Wasilko et al) streamlines the entire process from screening to large scale production, eliminating repeated time-consuming virus amplification steps. Baculovirus BIIC Banking provides a long-term stable storage form of recombinant baculovirus, particularly important for unstable virus stocks and for resupply of critical reagents over lengthy increments of time.

In this process insect cells are infected using a multiplicity of infection ratio (MOI) that would normally be used for expression (MOI= 1-5). The infected insect cells (Sf9 or Sf21) are harvested and frozen when the cell viability is high and the cell diameter has just started to increase. The cells are cryopreserved using standard controlled-freezing methods and the vials are stored in the vapor phase of liquid nitrogen. These densely packed recombinant baculovirus BIIC stocks when thawed can be used in screening or scale up, where as little as 1 mL of BIIC may be used to infect a 10L insect cell culture. This system reduces the need to produce and use 1L of high-titer, virus stock supernatants.

Baculovirus BIIC Banking Advantages:

Titerless System
Stable baculovirus stocks
Rapid, streamlined process
Readily scalable
Reduced time to scale-up

Keys to the Baculovirus BIIC Banking Process

Infect Sf9 cells with baculovirus

As the Multiplicity of Infection (MOI) increases, the percentages of cells infected with at least one viral particle also increases.

Plaque forming units (pfu) of virus used for infection

MOI = ________________________________________

number of cells.

In general, infected cells produce ~100 pfu/cell, therefore, it is important to estimate the MOI for the virus used with different cells

Allow the cells to become infected

This is a process that occurs over a few hours/ days and careful monitoring is required throughout the process. It is important to obtain infected but healthy cells. This is achieved by closely monitoring cell count, cell size and cell viability over time. In general, it is important to identify a cell diameter increase of ~2-3 μm and a viability of >90%.

Cryo-preserve the “healthy” infected cells

Baculovirus-infected insect cells (BIIC) are frozen using a standardized and effective method so that cell viability and infectivity on thawing are similar to the starting unfrozen materials.

The Kemp Baculovirus BIIC Banking Process:

Kemp Proteins transfect Sf9 cells and produce a P1 virus stock. The P1 virus stock is used to infect a sufficient amount of Sf9 cells to produce up to 100 BIIC vials containing a volume of 1 mL and ~1×10⁷infected cells. Monitoring at this stage is performed using both manual and automated methods:

- Human Microscopic review
- Virus Counter Plus (Sartorius)
- Cellometer Auto T4 (Nexcelom Bioscience)

A test expression is performed in Sf9 and Sf21 cells using one BIIC vial from step 1 using the standard BIIC infection/expression protocol.
Samples of culture supernatant and/or cell pellet are obtained at three time points and analyzed for protein expression by SDS PAGE and Western Blot using primary antibodies to standard affinity tags.
The BIIC vials can be shipped to the client or held at Kemp Proteins for use in future production scale-up.

NOTE: Client may supply protein specific antibodies for step 3 if required.

Client Supplied Materials: ≥5 ug of a pFastBac construct, miniprep of bacmid DNA

Kemp Proteins Deliverables: Frozen BIIC vials with Certificate of Analysis.

To learn more about Kemp Proteins: Baculovirus Expression Service

Ref: David J. Wasilko and S. Edward Lee. TIPS: Titerless Infected-Cells Preservation and Scale-up. BioProcessing Journal 5(3): 29-32. (2006).

Baculovirus BIIC Banking Quote Request

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