We deliver scalable and robust GMP-compliant processes for viral vectors.
The different upstream scales are matched with appropriately scaled downstream equipment for clarification, concentration, chromatography and filtration. For the purification of viral vectors, we offer clarification by depth filtration with a variety of different filters including filters that combine particle purification and impurity removal. Removal of nucleic acids is often required for viral vector products and can be achieved by techniques such as enzymatic digestion or fractional precipitation. Prior to purification, concentration and buffer exchange by tangential flow filtration can be performed using hollow fibers or flat screens. This is dependent on the physical properties of the target vector and the requirements of the final product.
Purification is normally achieved using several chromatography steps. The type of chromatography used is dependent on the vector properties. Ion-exchange or hydrophobic interaction differences are often exploited for purification, while size-based group separation or tangential flow filtration, be it hollow fibers or flat screens, can be used for polishing and formulation. Depending on the size of the viral product, a sterile filtration can be performed. For larger viruses for which this approach is not possible, we have extensive experience in validated aseptic manufacturing processes. For analysis of the product, we offer a variety of product specific assays and assay development services, such as TCID50, PFA, FFA, ELISA, (q)PCR and HPLC-based methods.
When developing a process from scratch, we start with selection of resins for binding and elution profiles, moving from 96-well plate screening to 1 mL spin traps and spin filters, to intermediate and large-scale purification columns using ÄKTA™ explorer, ÄKTA™ pure and ÄKTA™ pilot respectively. For viral vector purification, a two- to six-step process is usually required which results in 35-80% product recovery, thereby adhering to regulatory guidelines concerning purity.
When delivering research batches for pre-clinical studies we typically use centrifugation-based methods. For instance, when delivering adenoviral viral vectors, we use cesium chloride ultracentrifugation steps to obtain high purity vector preparations. For other vector systems, sucrose-based ultrafiltration or affinity purification can be used.
In general terms, a large scale viral vector purification process consists of clarification and concentration steps (sometimes preceded by cell lysis and DNA removal steps) followed by chromatography (charge, hydrophobic interaction, mixed mode, size exclusion, or affinity-based). Finally, the vector will be formulated by re-buffering and/or addition of components followed by a final membrane filtration step using a bioburden reduction or sterilizing grade filter. Although rare, in some cases we have experienced interference of the transgene product with the generic vector purification process and have successfully adapted these processes in response. In addition, we have experienced that different serotypes of viral vectors such as AAV or adenovirus may require substantial adjustment of the purification process to optimize the yield, as loading and elution conditions may vary considerably. This has also been described by Nass and co-workers for AAV vectors (Nass et al, 2017). To summarize, we have extensive experience in working with various vector platforms as well as specific product requirements.