Although the targets of biomanufacturing have evolved from therapeutic proteins to “advanced therapies” and now to gene therapies, the issues and solutions remain the same. The production of safe, effective medicines is achieved by means of controlled, well-characterized processes. So, while the therapeutic vehicle has changed (from a protein to a virus-delivered gene), the roles of process development and supporting analytics have not.

Today’s viral vector manufacturing faces much the same challenge that conventional protein biomanufacturing faced 30 years ago. Essentially, the challenge is to develop robust, well-characterized processing steps supported by rapid, reliable, high-throughput, and equally robust analytical methods.

The top consideration for any virus production process is ensuring consistent product quality through scaleup, says William Lee, PhD, research associate, Alexion Pharmaceuticals (a subsidiary of AstraZeneca). “Careful addition of the DNA complex to the bioreactor is one key scaleup factor that affects product quality,” he emphasizes. “We use advanced analytical methods, such as multiattribute long-read next-generation sequencing, and two separate potency assays to mitigate the potential for quality impact from modifying the upstream process.”

GeneCity facility in Turku, Finland
3PBIOVIAN is expanding its GeneCity facility in Turku, Finland. The facility, which will cover 69,000 square feet and support the production of AV and AAV products, is expected to be fully operational by 2025.

Process considerations

Switching adeno-associated virus (AAV) manufacturing from adherent-cell platforms to suspension-cell platforms has been a high priority. Suspension-cell systems are easier to treat, feed, characterize, and generally to work with, but the change requires a comprehensive comparability study. Developers typically adopt a risk-based, design-of-experiment approach to evaluate the independent effects of cell lines, media, enhancers, and harvest conditions—as well the potential impact of scale itself.

That is not to say successful adherent-cell processes are being phased out. 3PBIOVIAN, a viral vector contract development and manufacturing organization since 2004, has demonstrated the capability of its iCELLis adherent cell platform to scale up perfusion-mode production from 1.07 m2 scale to a production-capable 133 m2 bioreactor.

3PBIOVIAN developed its first adenovirus (AV) process more than 20 years ago, when interest in viral vector manufacturing was in its infancy. Since then, the company has supported around 15 commercial AV projects culminating in around 50 GMP drug substance and drug product batches. The iCELLis suite of services includes cell banking, seed stocks, process and analytical development, manufacturing, fill and finish, and final clinical trial batch release.

iCELLis takes a unique approach to perfusion bioreactors. “Unlike traditional suspension or microcarrier-based systems, iCELLis uses an adherent cell matrix, allowing for high-density cell cultures in a compact space,” says Antti Nieminen, 3PBIOVIAN’s group deputy CEO. “This system stands out by offering a scalable, automated system that works with various cell types, including CHO cells, which enhances its versatility in bioproduction.”

Scaling up is a common biomanufacturing challenge, mainly due to the need to maintain cell viability, productivity, and consistency at larger volumes. “iCELLis addresses these challenges by providing a controlled environment that supports high cell densities and efficient nutrient exchange,” Nieminen says. “[This approach leads] to robust and reproducible production outcomes.”

In AV production, the main challenge is obtaining high yields and high product purity at the same time. To overcome this challenge, iCELLis simplifies downstream processing.

iCELLis is applicable to perfusion-cultured HEK293 cells, another mainstay of both AV and AAV vector processing. In HEK293 cells, iCELLis improves cellular productivity by optimizing media and cell attachment, but these modifications, Nieminen says, may require careful optimization to avoid potential downsides such as altered cell behavior.

For example, Forge Biologics has developed a HEK293-based AAV production platform using the company’s HEK293 suspension Ignition Cells™ in single-use bioreactors.

“In our platform, process primary recovery involves harvest and clarification of the crude bulk material containing both full and empty AAV capsids,” says Frank K. Agbogbo, PhD, vp of process development at Forge.

company spokesperson Marina Corleto. “Contaminants, including empty vectors, residual plasmids, and process-related impurities (such as host cell DNA and RNA), are removed during downstream purification to yield a highly pure recombinant AAV appropriate for clinical use.”

Clarified harvest material is further processed through affinity chromatography, full particle enrichment, and final formulation.

Forge Biologics facility, the Hearth, in Columbus, OH
Forge Biologics has built a 200,000-square-foot facility, the Hearth, in Columbus, OH, to provide plasmid and AAV manufacturing services. It houses 20 custom-designed cGMP suites with 200,000 L of manufacturing capacity.

Analytical support

By now, every bioprocessing organization recognizes the key ingredient to navigating new production technology. “There is an increasing demand for rapid, reliable, high-throughput analytic methods to facilitate process development for viral vectors,” says Srivatsan Ramesh, PhD, downstream process development scientist, BridgeBio Pharma.

BridgeBio fills that need with a suite of “straightforward yet robust” analytical methods for development- or manufacturing-stage viral vectors. The company’s approach involves quantifying capsid titers using ELISA and biolayer interferometry, quantifying vector genomes with droplet digital PCR, and identifying the genome of interest, followed by potency assays, particle size distribution and aggregate analyses, and assessments of product- and process-related impurities.

According to Ramesh, “continuous improvement” will occur through implementation of new analytical methodologies for determining particle size distribution, aggregation, and concentrations. For example, assessments of multiple-angle light scattering in-line ultraviolet absorbance can be used to quantify vector concentrations. The company has adopted a similar analytic strategy to support screening for bioreactor conditions, resins, and buffer systems. This approach accelerates process development, Ramesh says, while improving bioreactor productivity, reducing downstream impurities, and enhancing process efficiencies.

Process analytic technology (PAT) implementations have been progressing in the manufacture of AAVs, albeit slowly, as slowly as PAT implementations have been progressing in the manufacture of therapeutic proteins. “Considering the novelty of AAV bioprocessing, the adaptation of PAT in downstream biopurification has been inching forward rather slowly,” Ramesh says. “Our progress in this area has involved the implementation of online characterization using UV-visible spectrometry, and rapid, high-throughput capsid quantification using biolayer interferometry. Continued advancements in materials research for improved sample capture, separation, and signal attenuation will be required for the specific needs of AAV manufacturing and PAT development.”

Beware the empty capsid

One major recurring challenge in viral vector downstream processing is removing empty capsids while ensuring drug substance homogeneity, consistency, and stability. “The high abundance of empty capsids is a major challenge during production,” says Ohnmar Khanal, PhD, downstream technology lead for downstream purification, Spark Therapeutics. “Since empty capsids lack the disease-treating transgene, they increase the viral load in the drug product without increasing the clinical dose, which is typically specified based on transgene titer.”

In other words, empty capsids provide all the potential drawbacks of treatment—for example capsid-triggered immune responses—with none of the benefits. Furthermore, from a processing perspective, carrying a large percentage of empty capsids from the bioreactor through the capture step is expensive and resource intensive. “That is why decreasing the empty/full ratio is highly desirable in both upstream and downstream AAV process development,” Khanal explains.

Empty capsids are usually removed chromatographically, but many factors come into play, for example, the effects of resin geometry, chemistry, kosmotropic buffer agents, and metal ions. Spark Therapeutics has developed a process that it claims removes more than 90% of empty capsids, with an overall yield of 80%.

Empty capsids are similar chemically and structurally and were all thought to be identical, but that turns out not to be true—which complicates their removal. Since they are chemically and structurally similar, though, removing them chromatographically can be challenging, especially with ion exchange (polishing) purification methods.

Spark’s approach to empty capsid removal involves first understanding empty capsid protein heterogeneity, then devising a combinatorial method that targets the various populations of empty capsids to achieve their removal to greater than 90%.

“Since empty capsids can degrade on the column, we developed a proprietary approach through which only full capsids bind to the polishing chromatography resin,” Khanal relates. “These may then be recovered after the empty capsids pass through and are discarded.”

Spark Therapeutics Gene Therapy Innovation Center
Spark Therapeutics is building its Gene Therapy Innovation Center, a 500,000-square-foot, multistory facility on Drexel University’s campus in Philadelphia, PA. It will serve will serve as a Roche center of excellence for gene therapy manufacturing globally.

A typical process

Forge Biologics, a gene therapy contract development and manufacturing organization, has developed an efficient, scalable, cGMP-worthy process for producing FBX-101, a recombinant AAV expressing galactocerebrosidase (GALC). FBX-101 is a treatment for Krabbe disease.

FBX-101 instructs the patient’s cells to produce a functioning GALC protein. The FBX-101 capsid delivering the GALC transgene targets all cells in the body but has enhanced tropism for the peripheral nervous system. This approach reduces process development time and costs, at up to 1,000 L scale, for clinical-grade materials.

The Forge Biologics platform process involves transient transfection using the company’s Ignition HEK293 suspension cells in single-use bioreactors to generate FBX-101 suitable for clinical use. “Process scalability has been achieved with the alignment of power per unit volume for agitation, volumetric gas flows, and kLa for gas sparge rates from 250 mL to 1,000 L,” explains Frank K. Agbogbo, PhD, vice president, process development, Forge Biologics.

“In our platform process, primary recovery involves harvest and clarification of the crude bulk material obtained from the optimized upstream production process containing both full and empty recombinant AAV capsids. The process contaminants—empty vectors, residual plasmids, and process-related impurities such as host cell DNA and RNA—are removed during downstream purification unit operations to yield a highly pure recombinant AAV appropriate for clinical use.”

The clarified harvest material is further processed through affinity chromatography, full particle enrichment, and final formulation. The recombinant AAV vectors produced from shake flasks and bioreactors at the different scales were compared for titer (by droplet digital PCR), residual plasmid (by droplet digital PCR), residual host cell protein, purity (by SDS-PAGE), genome integrity (by alkaline gel electrophoresis), empty/full particle ratio, and potency.

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