Guiding Principles In Process Development For A Viral Vector

At Batavia Biosciences, we adhere to three guiding principles in developing processes for viral vector products;

1. First Time Right
2. Cost-Efficiency
3. Bullet-Proof Documentation

These principles are briefly outlined below

1.  Get Process Development Right The First Time

Developing a production protocol that allows smooth entry into phase-I and phase-II clinical trials starts with a thorough understanding of the expected commercial manufacturing process. Thus, all raw materials, equipment, and process architecture designed or developed for clinical manufacturing must be seamlessly extrapolated to a commercial scale. Should this not be taken into account, there is a severe risk that a process will need to be substantially re-designed at a later stage, which can trigger regulatory authorities to demand new clinical trials to demonstrate safety and efficacy. We have a long-standing track record in building detailed and realistic product development plans for various products, including many viral vector-based products. Such a plan provides a road map to help our customers’ management choose the proper path in developing the process and all other steps involved in bringing the product to the patient. 

Getting it right the first time is critical to ensure building shareholder value. In addition, it is vital in the viral vector field when markets, patient populations, and dosing regimens for many of these types of products are constantly in flux.

To quote Yogi Berra (American Baseball player):

“If you don’t know where you’re going…
You will end up someplace else”.

2. Realizing Cost-efficient Viral Vector Production

Manufacturing costs are one of the main cost drivers for a product. Therefore, it is essential to establish a cost-efficient manufacturing process. To gain control over this process, when designing manufacturing processes, high priority is given to:

• The efficient pre-culture process allows optimal use of the production bioreactor
• Optimal process yield obtained in bioreactors (USP)
• Optimal purification recovery (DSP)

These are all critical drivers of the Cost of Goods. All these drivers must be carefully evaluated so that optimal output parameters and acceptable bandwidths are known. These parameters will then be used to determine success or failure during process development (design space). We offer  in silico  cost modeling  services and have a long-standing track record in developing processes aligned with pivotal economic drivers. We have amassed a portfolio of  technologies  and capabilities that aid the reduction of the Costs of Goods and development timelines.

To quote Lucius Seneca (Roman philosopher):

“Luck is a matter of preparation meeting opportunity.”

3. Aim For Bullet-Proof Documentation

At Batavia Biosciences, the QA department has been involved in R&D project discussions as a pivotal resource from day one. Without a doubt, this is a significant contributing factor to our success in IND and IMPD support. To demonstrate the importance of early QA involvement, we share three early QA initiatives that are drivers of ensuring the fastest route to the clinic.

Planning phase: Without exception, every customer wants to know how to shorten the time to the clinic or when investments in product development can be postponed. Our QA department assists the technical staff and the customer in developing a detailed product development plan, in which risk-based decisions on the optimal path to the clinic are justified, laid down and locked in.

One Quality Management system: In the R&D environment, all selected materials and equipment are suitable for GMP manufacturing. In this way, the developed process is directly transferable to a GMP facility without the need to repeat any parts of the final process. In addition, the Quality Management system used in R&D is identical to that used in GMP.

Avoid internal tech transfer: Our laboratory staff members working in R&D are also trained and qualified to work in a GMP environment. Therefore, the same team that develops the production and purification processes in R&D is also fully trained and able to take the project into GMP. In this way, we avoid time-consuming internal technology transfer processes that would otherwise be required.

To quote Walter Disney (American film producer):

“Always fight for quality… whether giving or receiving.”

 

Upstream Process Development For Viral Vectors

We operate various manufacturing scales and utilize diverse cell culture equipment for adherent and suspension growth. The scale ranges from high-throughput 20 mL spin tubes to 200 L bioreactors. In addition, the equipment ranges from mini bioreactors, shake flasks, WAVE Bioreactors™ , and various stirred tank bioreactors to fixed-bed bioreactors (iCELLis® and scale-X™ systems) to support high cell density cell cultures.

Product yields for viral vectors

We perform state-of-the-art manufacturing of diverse viral vectors on par with literature values. For instance, typical yields of 1013 virus particles per liter cell culture of replication-deficient adenoviral vectors are obtained (Vellinga et al, 2014). Typically, 105 viral genomes per cell can be obtained for AAV vectors (Aponte-Ubillus, 2018), and 107 transducing units per milliliter of culture for lentiviral vectors (Merten et al, 2017). For measles vectors, typically 107 TCID50 units/mL are obtained (Grein et al, 2016), whereas for VSV vectors typical titers obtained are 108 plaque forming units per milliliter (Clarke et al, 2016). Here, it must be noted that these reported yields do not consider variations in transgenes, promotors, production equipment, cell line platforms, and cell and virus culture media. Therefore, yields from bioreactor harvests may vary from product to product.

Upstream process development tools

We utilize Design of Experiments, our generic manufacturing protocols, and our SCOUT® technology for product-specific protocol development. SCOUT technology can be used, to test a large panel of production media and generic feed strategies. In a SCOUT experiment, cultures are scaled down in mini bioreactors to test various growth conditions to optimize cell growth efficiently. The critical deliverables are short cell population doubling times, high cell density and viability. In addition, the SCOUT technology is used to rapidly anchor critical parameters such as multiplicity of infection (MOI), time of harvest and best plasmid transfection practices. Findings from the small-scale studies will subsequently be translated into pilot and large-scale protocols to establish the manufacturing process further. 

We have generic standard operation procedures (SOPs) available on vector production for all our viral vector systems, which can be used to develop the product-specific production process.

 

Downstream Process Development For Viral Vectors

The different upstream process formats and 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 various filters. This includes filters that combine particle purification and impurity removal. Removal of nucleic acids is often required for viral vector products. This removal can be achieved through enzymatic digestion or fractional precipitation. Before purification, concentration and buffer exchange by tangential flow filtration (TFF) can be performed using hollow fibers or flat screens. This depends on the target vector’s physical properties and the final product’s requirements.

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 TFF 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 product analysis, we offer various product-specific assays and assay development services, such as TCID50, PFA, FFA, ELISA, (q)PCR and HPLC-based methods.

Downstream processing equipment

When developing a process from scratch, we start with a selection of resins for binding and elution profiles. We then move 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, the aim is to limit the number of chromatography steps resulting in high product recovery while adhering to regulatory guidelines concerning purity. We’ve experience setting up two to six-step processes, resulting in an overall downstream yield of 35-80%.

Small-scale viral vector purification

When delivering research batches for preclinical studies, we typically use centrifugation-based methods. For instance, we use caesium chloride ultracentrifugation steps to deliver adenoviral viral vectors to obtain high-purity vector preparations. For other vector systems, sucrose-based ultrafiltration or affinity purification can be used.

Large-scale viral vector purification

Generally, large-scale viral vector purification processes consist 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 adding components. This is 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. Nass and co-workers have also described this for AAV vectors (Nass et al, 2017). To summarize, we have extensive experience working with various vector platforms and specific product requirements.

Read more in our blog about the bottleneck in downstream processing of viral vectors.

Analytical Development For Viral Vectors

The development of innovative viral vector manufacturing processes strongly depends on the availability of accurate and reproducible product characterization and release assays. Release tests for viral vector products typically include tests to measure quantity, potency, vector genetic stability, identity, residuals (i.e. host cell DNA, host cell protein and benzonase), and safety.

The product needs to be characterized to determine which parameters are essential to monitoring during process development and which are necessary for release. Most of the techniques and assays that are used for product characterization are performed in-house. Some of these assays are developed to support process development, using representative material, and then validated for release of the final product. Our expert analytical staff covers all aspects, from design to implementation and development to validation of the assays according to ICH guidelines. Some compendial and most biosafety assays are outsourced to our qualified partners. For outsourcing, we can provide support across all areas, including the selection of partners, quotation, temperature-controlled shipments, review and reporting in certificates of analysis.

Controlling materials for viral vector production

Proper control must be established over the materials used to construct and manufacture viral vectors. The use of animal-derived components is avoided as much as possible. When unavoidable, traceability and testing must be in place to mitigate any risk of introducing and transferring extraneous agents in the process. Extensive testing- by PCR for in vitro and in vivo adventitious agents- to confirm the absence of adventitious viruses in master cell banks (MCB) and master virus seeds (MVS) is performed according to ICH Q5A and ICH Q5D guidelines.

Genetic stability testing

Another important parameter in monitoring the production of viral vectors is genetic stability testing. Here, the vector is propagated for several passages until or beyond the envisioned commercial manufacturing stage. Analysis of the transgene region is needed to maintain the proper antigen expression. Extended propagation of the starting material provides the sensitivity required to detect recombinants or mutants that have gained a growth advantage over the target vector. PCR detection combined with sequence analysis provides sufficient specificity and sensitivity to detect any variants in the transgene region that might arise. Next-generation sequencing technologies can be employed to characterize vector variants that may be present at low frequencies in virus seeds.

Analytical development expertise

We have ample experience developing and qualifying a broad range of assays, including (q)PCR, cell-based assays (FFA, PFA and TCID50), HPLC, SDS-PAGE, western blot, and ELISA. As such, we deploy a full range of assays required to monitor the development and release of viral vector-based products successfully.

 

In Silico Cost Modeling For Viral Vectors

A robust, cost-effective, and high-yielding manufacturing process will support your product’s financial viability when developing a viral vector product, such as vectored vaccines, gene therapies or oncolytic viruses.

Unfortunately, we see product developers are regularly confronted with the cost implications of unexpected changes during scale-up for clinical manufacturing. In silico cost modeling of manufacturing processes provides insights into the Cost-of-Goods of the production process so that you can make informed decisions.

The  in silico  modeling of production processes allows product developers to gain insight into the main cost drivers of the process in an early development stage. This helps you to make well-informed decisions in the design of manufacturing processes quickly. It saves time and resources during the  development  phase and will lead to lower Cost of Goods during the  manufacturing  of viral vectors. For the cost modeling, we use the BioSolve Process software package, developed and marketed by  Biopharm Services. This software package integrates the latest industry-benchmarked price information for labor, materials, consumables, and equipment into the modelled process, leading to up-to-date insights.

Benefits of in silico  cost modeling

In silico cost modeling is a key tool for understanding cost implications of technology and process choices. It allows the design of the most cost-effective process set-up and the selection of optimized technologies supporting the process under development. Thus, it tremendously contributes to the long-term planning of product developers. In silico modeling helps to reduce risks by quickly carrying out multiple process comparison analyses and facility utilization assessments. We use the BioSolve Process software package to help develop business cases for novel and innovative technologies or process options. With the many analysis tools available, we perform analyses of multiple process comparisons before presenting our optimized, most cost-effective solution. Next to using in silico modeling for novel processes, we have also successfully deployed the system to optimize existing biopharmaceutical manufacturing platforms. Our in silico modeling services provide our customers and us with the opportunity to:

• Assess the manufacturing process at a commercial scale, during early development phases
• Determine whether a process will fit in preset facility designs
• Compare process choices and technologies from a cost perspective
• Plot technology options to identify the best fit for meeting production targets
• Configure any viral vector process on upstream processing and downstream processing
• Design improved process technologies

With our in silico modeling expertise, we can reduce manufacturing costs by understanding cost structures early in the development of non-platform and platform processes.