Downstream Process Development of Viral Vectors

The role of the downstream process is to enrich the viral vector product while eliminating contaminants from the host cells and media. Creative Biogene leverages its expertise and experience to bring solutions to the unique challenges faced in the downstream processing of viral vectors, ensuring the continued scale-up and optimization of key production processes.

Challenges in Downstream Purification of Viral Vectors

Viral vectors as new therapeutic modalities are complex vectors with their own unique production and purification challenges. The following difficulties may arise in downstream purification due to the choice of vector type or upstream platform and process:

The vector supernatant produced by transfection contains a large amount of contaminating plasmid DNA.
Use of suspended cell bioreactors generates more debris than fixed bed bioreactors.
Large viruses, such as herpes, may not be filtered for decontamination and may require aseptic processing, and the attendant validation. The use of cell culture media supplemented with serum can also result in a greater purification burden, and increased serum viscosity can affect downstream process efficiency.

Although there are many different methods to purify viral vectors, most of these existing processes have drawbacks. They either lack the scalability needed to purify large numbers of vectors or require many inefficient steps that are too costly and less productive due to cumulative yield loss.

Downstream Process Development Strategy for Viral Vectors at Creative Biogene

The downstream process for viral vectors involves several steps including lysis, clarification and purification of the viral vector. The choice of purification method further depends on factors such as cell line and virus properties, as well as harvesting method and volume. Creative Biogene offers customized processes for different viruses and their production methods to help clients overcome challenges in downstream processes.

Downstream Process Development Strategy for Viral Vectors at Creative Biogene

Lysis step

Cell lysis can be performed by chemical methods (e.g. decontaminant agents or hypertonic solutions) or physical methods (e.g. multiple freeze-thaw cycles, microfluidization or sonication). Typically, we favor the use of chemical lysis methods to scale up viral vector production. The cell lysis step is usually followed by an enzymatic treatment step to degrade the released nucleic acids, followed by a subsequent purification step.

Clarification step

To minimize impurity and particle challenges prior to subsequent chromatography unit operations, Creative Biogene removes process-related impurities through the use of advanced deep filtration systems. Our commonly used Tangential Flow Filtration (TFF) has proven to be an effective method of clarification and concentration with recoveries as high as 90-100% and minimizing shear throughout the purification process, increasing yields.

Purification step

Creative Biogene efficiently isolates intact viral particles through a two-step purification process that commonly includes ion exchange or affinity chromatography. These methods offer the flexibility of process scale-up for both laboratory and scale up production and are suitable for the removal of multiple impurities such as DNA, proteins and viruses.

Advanced Methods for Downstream Processes

Method	Description
Chromatography	Chromatography is widely used and can be applied to a variety of downstream steps, including capture, concentration, purification and refinement steps.
Affinity chromatography (AC)	AC relies on the interaction between viral particles and a ligand that has a high affinity for the target, but not for the contaminant.
Ion exchange chromatography (IEC)	IEC is a simple, versatile and cost-effective technique that has become a key step in the purification of many viral vectors. IEC can be used in binding/elution mode (e.g., virus capture or HCP removal) or in flow-through mode (e.g., DNA removal), and it has been found to be important for both DNA and HCP removal.
Size exclusion chromatography (SEC)	SEC can be used to separate viral particles from contaminants based on size and shape. the advantage of SEC is that it is very gentle on particles because it has no binding or elution and is performed isometrically.
Tangential flow filtration (TFF)	TFF technology can be used in multiple locations throughout the downstream process. It has shown good scalability, process economy, process robustness, and in many cases can provide yields near or above 90% compared to alternative technologies such as centrifugation.

As an experienced provider of viral vector CDMO services, Creative Bionene has a clear understanding of the factors that have a significant impact on downstream processes. We are committed to overcoming downstream process challenges and bringing cost-effective solutions to our clients to facilitate the development of viral vector-related therapies. Please feel free to contact us if you have a need, and our experienced specialists will be happy to assist you.

* For research use only. Not intended for any clinical use.

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