NC-VVIRAL features novel affinity adsorbents that accomplish:

• One-step purification and the full-capsid enrichment of Adeno-Associated Viruses (AAVs)
• Isolation of transducing Lentiviral Vectors (LVVs)
• Purification of Adenovirus and Baculovirus
• Purification of Virus-like Particles (VLPs)

We employed a scalable rAAV production process using our Pinnacle PCL platform that reduces the high costs associated with transfection-based processes. However, at cell densities that yield high volumetric productivity, the increased impurity burden prohibits the practical use of conventional filtration schemes. Through optimization of our filtration scheme, we have designed a robust downstream process capable of handling the challenging feed stream of a high-cell density production.

ASO-based therapies have offered effective treatments for many neurodegenerative diseases through addressing the pathology as opposed to the symptoms. Next-generation ASO therapies via new chemistries pose even greater clinical promise (e.g., longer duration), though they can disrupt existing ASO processing platforms. This presentation summarizes the purification of a late-stage, next-generation oligonucleotide that encountered, and later overcame, several challenges to deliver a drug substance of expected high purity and yield. 

Non-mAb proteins are known for their complex structure, poor expression titer, prone to aggregation, and sensitivity to process stresses. These modality related issues often complicate the downstream processes and compromise their performance. This presentation discusses the common challenges and strategies to improve the non-mAb harvest recovery, streamline the chromatography layout and operations, enhance the process effectiveness for viral and HCP clearance, and minimize product and process impurities in the bulk.

This presentation details the platform development of a purification process for a new class of non-viral gene therapies. The talk will emphasize the optimization of initial steps including harvesting, lysis, and clarification.

This work highlights the development of a redox reaction that occurs during the capture chromatography step resulting in the efficient formation of multispecific antibodies. The method consists of simultaneously binding two separate homodimers to a chromatography resin then applying a reductant wash to reduce the interchain disulfide bonds in both antibodies. The antibodies are then eluted and neutralized in the presence of an oxidant to form the heterodimer. During this work, the mechanism and kinetics of reduction, heterodimerization, and oxidation was characterized to maximize conversion and product quality.

Buffer requirements for large-scale purifications present significant facility storage and resource demands. Buffer concentrates, which minimize these constraints, have been successfully implemented in thirteen 500 L pilot production runs at Alexion, AstraZeneca RDU. An inline dilution system has produced buffers within tight tolerances as measured by offline pH, conductivity, and density measurements. The system has reduced buffer volumes by 50%, preparation time by 66%, and storage space by 50%. Usage of the system has also had an added benefit of reduction of consumables, thus helping to lower costs and meet sustainability goals.

ICH Q5A was recently updated, which addresses viral safety of biotechnology products derived from cell lines of human or animal origin, and the updates will be discussed along with best practices to address and manage the changes within CMC programs. In addition, updates to Annex 1 and how it applies to downstream operations will be presented.

Self-removing affinity tags provide a powerful platform for purifying untagged recombinant proteins without the need for proteolytic tag removal and have been successfully applied to a variety of biosimilars and other therapeutic protein classes. This work focuses on methods for validation of tag removal from the purified product as part of a cGMP manufacturing platform. Several case studies will be provided, with specific steps and data provided.

Purification of large biomolecules and bioparticles, including large plasmids, virus, virus-like-particles, and vesicles, by flow-through chromatography has been made practical with the availability of effective core-shell resins. We examine the structural and functional properties of commercial agarose-based core-shell resins and develop models to describe the kinetics of binding for proteins with a broad range of molecular mass in single and multiple component systems and predict the dynamic binding capacity.

Multicolumn chromatography is an established strategy to improve productivity and reduce resin usage. However, the increased column loading can lead to elevated levels of impurities, resulting in a trade-off between productivity and product quality. In this presentation we describe different strategies, including harvest improvements, column loading optimization, and wash condition screening, to mitigate this challenge and improve impurity clearance while maintaining comparable product quality and process performance to batch processing.

Depth filtration is routinely used for primary clarification of cell culture fluid, but its analysis and design are almost entirely empirical. We present a conceptual mechanistic model that can account for sieving, adsorption, and caking in modeling the pressure drop and filtrate turbidity, and also use multiple experimental methods to obtain supporting data to aid in model discrimination regarding the mechanisms involved.

Advances in cell culture processing have not only resulted in increased cell densities and productivity, but also in increased levels of solids and sub-micron particles, which decrease the efficiency of the cell separation step through centrifugation and depth filtration. A harvest method using a cationic polymer, namely pDADMAC, was investigated for the removal of colloids and for improvements in cell clarification. This presentation will focus on the implementation of large-scale harvest using pDADMAC flocculation and examination of pDADMAC flocculation performance using small-scale studies.

Affinity Capture is the preferred method for primary capture in biotherapeutic downstream processing. However, the hash elution condition may be incompatible with product stability and negatively impact product quality. We propose a novel approach to the design of affinity elution buffers for challenging products by leveraging our new discovery, which could maintain product quality, achieve high yield, and assure maximum compatibility with subsequent step.