Q&A Session: Development And Application Of Insect Baculovirus System in Gene Therapy Viral Vectors, Proteins And Vaccines

Panda Classroom launched the CGT Lecture Series in OctoberIn October, Panda Classroom launched the CGT Lecture Series, aiming to provide a learning and exchange platform for academia and industry in the CGT field.

In the previous session, we invited Dr. Xing Pan, R&D Director of GeneFerm Therapeutics, to Panda Classroom. She delivered a presentation themed "Development and Application of the Insect Baculovirus System in Gene Therapy Viral Vectors, Proteins and Vaccines", where she elaborated on the application of the insect baculovirus system in large-scale AAV production, as well as in the fields of protein and vaccine development. Dr. Pan also introduced optimization strategies for the Bac/Sf9 platform from multiple perspectives, including plasmid design, medium screening, upstream cell culture processes, and downstream purification processes.

 

During the live broadcast, participants showed great enthusiasm for learning and raised numerous questions. Due to time constraints, not all questions could be answered during the session. A summary of the questions and answers is provided below:

 

Q: Regarding cell thawing for resuscitation – to avoid ice crystal formation that damages cell walls, it is recommended to thaw the cells completely within one minute. However, some researchers suggest thawing the cells only halfway, then using 37℃ medium to dissolve the remaining frozen solution. This method of incomplete thawing in the water bath is said to avoid the cytotoxicity of DMSO at 37℃. Both approaches seem reasonable; how should we choose between them?

A: Generally speaking, rapid thawing to prevent ice crystal formation is the more conventional method. It is recommended to thaw the cryovial in a water bath until only a small ice crystal (about the size of a mung bean) remains. However, if your cells are highly sensitive to DMSO, the second method may be more suitable. Additionally, you can opt for a professional cell resuscitation instrument.

 

Q: For adaptive culture of cells in a new medium, how many passages are required to observe definitive results?

A: Generally, at least 3 passages are needed, but there can be significant variations among different cell types. It is advisable to cryopreserve cells after each passage. If you have further questions about cell suspension domestication or adaptation to new media, please contact us.

 

Q: In large-scale AAV production using insect cells, how does the selection of upstream processes differ for various AAV serotypes?

A: For the One-Bac 4.0 system, GeneFerm has established a comprehensive AAV platform process. For different serotypes, adjustments to upstream Critical Process Parameters (CPPs) are made based on the AAV's Critical Quality Attribute (CQA) test results, such as the selection of batch/fed-batch mode and MOI (Multiplicity of Infection) strategies.

 

Q: Could you provide more details about the fed-batch process? How is the feeding strategy determined?

A: Feeding strategy is influenced by factors including feeding volume, feeding timing, frequency, and cell density. It is generally designed based on cell metabolism data and through multi-factor Design of Experiments (DOE). (For more details on process optimization, please refer to the replay of the live broadcast.)

 

Q: When producing influenza vaccines using the insect baculovirus system, which antigens are typically selected?

A: You can choose either HA (Hemagglutinin) or VLPs (Virus-Like Particles).

 

Q: Are the Sf9 cells from GeneFerm Therapeutics free of rhabdoviruses?

A: Yes, they are rhabdovirus-free.

 

Q: How can we optimize the heterogeneous glycosylation of influenza antigens expressed in insect cells?

A: You can try modifying fermentation conditions. Additionally, some literature reports suggest adopting genetic engineering approaches, such as knocking out or knocking in specific glycosyltransferases to alter the glycosylation pattern of insect cells.

 

Q: Post-translational modifications, particularly glycosylation, can affect the efficacy of antibody drugs. You mentioned that proteins produced by the insect platform lack terminal sialylation. Could other protein modifications impact antigen quality, and how can we control them?

A: Different expression systems lead to distinct protein modifications. For example, prokaryotic expression systems (e.g., E. coli) cannot perform glycosylation or proper protein folding, making them unsuitable for expressing complex proteins. In the early stage of any project, it is essential to evaluate the differences between various expression systems to select the most appropriate one. Alternatively, you can entrust GeneFerm Therapeutics to test protein expression across different systems.

 

Q: Why do recombinant proteins expressed by baculovirus form inclusion bodies? How can we improve protein solubility?

A: Inclusion body formation can be caused by factors such as rapid, high-level protein expression within a short period and intrinsically poor protein solubility. To enhance solubility, you can optimize the promoter, use solubility-enhancing tags, or test different signal peptides.

 

Q: For transmembrane protein expression, how can we ensure complete cell lysis to obtain recombinant proteins from the cell membrane?

A: For transmembrane proteins, it may be necessary to use different cell lysis conditions to ensure thorough lysis. Examples include using RIPA lysis buffer, RIPA buffer combined with sonication, or specialized transmembrane protein extraction reagents.

 

Q: The yield of recombinant proteins expressed using the insect baculovirus system is very low (less than 5 mg/L). How can we increase the production yield?

A: You can select a suitable promoter, perform codon optimization, and optimize fermentation processes.

 

Q: What is the typical expression yield of circular proteins in insect cells?

A: Currently, the initial fermentation yield of circular proteins in the industry is at least 100 mg/L.