To develop vaccine manufacturing processes more efficiently and quickly, the use of advanced process control, automation, and PAT are essential. With these levers, the end-to-end drug substance cycle was reduced from 2-3 weeks to only 1 week: starting with a seed vial on Monday and ending with drug substance bulk on Friday. Each unit operation was tailored to accelerate the entire drug substance cycle. The chain of unit operations was structured so that manual and semi-automated steps occurred during normal business hours and fully automated unit operations occurred overnight. In-line PAT enabled advanced process control and real-time release. End-to-end fully automated fermentation required no manual interventions between inoculation and harvest. Unit operations for buffer exchange and concentration occurred overnight, as needed, with tangential flow filtration. In-line total protein was measured throughout clarification and purification and used for advanced process control and real-time release. The required advances in process control, automation, and PAT elements that achieved this remarkable reduction in drug substance cycle time are not cost prohibitive and can be used in other industries and applications. Reduction of drug substance cycle time has wide ranging application in the manufacture of microbial enzymes and proteins, vaccines, and other biologics.

An anion exchange-based capture chromatography step for pDNA purification was developed and optimized at small (5mL) scale on the AKTA Avant system. Different chromatography supports were evaluated for clearance of host cell impurities, including host cell RNA. In addition, buffer solutions were optimized to maximize pDNA purity. Finally, the optimized process was scaled up to manufacturing scale using the AKTA Process skid (30L load). Minor changes to the process were required at scale in order to compensate for pressure limits on the chromatography skid, and the resulting process successfully replicated small-scale results in terms of yield and purity.

The old physical problem worsens with the large new modalities such as viral gene therapy vectors, where binding capacity is lost due to required channel or pore size. Rules will be presented to find the best compromise with beads, fibers, and monoliths. The biophysical properties relevant to adsorption and resolution of chromatography will be compared to antibodies and an outlook will be given on whether a platform process is possible.

We present a micro-chromatographic device for rapid development of methods for protein and gene therapy purification. The device houses multiple parallel columns and a dilution architecture that provides independent control of (1) load ratio, (2) formulation of binding, washing, and elution buffers, and (3) format of the elution step. The device is connected online to UV/fluorescence spectrophotometry and light-scattering detectors. Studies on mAb and viral vector purification are discussed.

Cation Exchange Chromatography (CEX) is a typical step in downstream processes for therapeutic proteins as it plays a significant role in clearance of process and product related impurities. In this case study, we have screened various commercially available CEX resins to find a high-capacity resin to improve operational efficiency, facility fit and reduce the cycles of resin usage to minimize the cost of goods. We assessed the static binding capacity (SBC) for different resins to identify buffer systems with varying pH and salt conditions which provide the maximum binding capacity. Based on optimal conditions found in SBC studies, dynamic binding capacity (DBC) studies were performed at bench-scale to identify the high binding resins. Following DBC studies, bench-scale runs were performed to evaluate the impact on process performance and product quality. One of the assessed resins demonstrated a higher binding capacity with higher purity and acceptable yield for a better facility fit.

Virus-like particles (VLPs) are promising for nanobiotechnology and vaccine development due to their structural similarity to viruses and strong immunogenicity. We previously developed a nickel-based affinity aggregation method for VLP purification. Here, we explore alternative metals (zinc, calcium, iron, copper, cobalt) for metal-ion affinity aggregation, achieving >50% recovery and >90% purity of turnip yellow mosaic virus coat (TYMVc) VLPs in Escherichia coli. Using DLS, TEM, and ITC, we characterize capsid formation and metal-histidine interactions. This study provides insights into optimizing low-toxicity metal alternatives, improving purification efficiency, and advancing sustainable, scalable VLP production for biopharmaceutical applications.

To develop a process control strategy for the production of safe and effective biologics, experiments are performed at small scale to provide operating ranges to the manufacturing site. For those ranges to be valid, the small-scale model must be capable of predicting manufacturing-scale performance. In this case study, we share a challenge encountered during the qualification of a small-scale UF/DF, describe the subsequent root cause investigation, and explain how the model was updated to ensure it was fit for purpose.

A novel class of peptide affinity ligands was developed to enable selective purification of capped mRNA transcripts based on 5′ cap recognition. Lead peptides were discovered through rational design and high-throughput screening, and validated via microarray, competitive binding, and AFM. In parallel, peptides targeting 5′ triphosphate structures were identified to remove uncapped byproducts, supporting a cap-specific strategy to enhance mRNA purity and therapeutic quality.

An overview of different ADC platforms used at Byondis will be presented together with challenges encountered and solutions found during development and processing. Challenges to control heterogeneity will be discussed and strategies for ADC manufacturing will be presented, as well as strategies for improvement of future processes.

As the ADC landscape becomes more complex, FDRIs can become increasingly difficult to remove through UF/DF. As a result, scientists have been relying on other traditional biologics methods for purification such as chromatography to achieve this task. This can add significant cost and time to the ADC production. This presentation focuses on a novel purification that utilizes activated carbon for removal of FDRI to change the model for ADC purification.

Topics being considered for discussion on virus reduction during the bioprocess include: 1) chemical (e.g., detergent), physical (e.g., temperature, filtration) and combined procedures, 2) traditional and novel technologies for measuring virus concentration, and 3) selection of virus model systems for validating log removal of viruses.

• Rethinking purification: How metal-induced aggregation simplifies protein recovery by reducing reliance on chromatography.
• Efficiency vs purity: Comparing aggregation-based methods with traditional techniques like IMAC. Where does it excel, and what are the trade-offs?
• Scalability & industrial applications: Can this approach be adapted for large-scale bioprocessing and high-yield protein recovery?
• Future perspectives: What advancements are needed to make metal-induced aggregation a mainstream purification strategy?
  

Bispecific antibodies are highly aggregation prone and difficult to be removed. This presentation will discuss effective ways to remove aggregates in both bind-elute and flow-through modes chromatography. We will also demonstrate how to mitigate chromatography induced aggregation by mixed-mode chromatography.

Significant aggregation challenges were encountered during the manufacturing processing of an immunocytokine diabody fusion protein. Emphaze AEX Hybrid Purifier, a fully synthetic quaternary amine functionalized anion exchange (AEX) nonwoven filter, was found to be particularly effective in removing aggregates. Pre-filters were identified to improve Emphaze filter throughput and further enhance aggregate reduction during the harvest clarification. The effect of operating pH and feed stream aggregate level were evaluated for the post-Protein A intermediate filtration and the aggregate binding capacity of the Emphaze filter was assessed. The study also elucidated the aggregate binding mechanism, which was primarily attributed to electrostatic interaction.

This presentation will demonstrate that the developed process is scalable with high yield, high efficiency, and robust biosafety, and that the purified product meets product safety, quality, identity, potency for phase I clinical support. Some challenges and solutions associated with process capacity, impurity removal, and biosafety will be discussed.

This talk will address the unique challenges in developing effective virus filtration strategies for rAAV gene therapy products. We’ll examine the role of virus filtration in enhancing viral clearance robustness and its increasing regulatory emphasis in AAV manufacturing. Key topics include evaluating commercially available virus filters for AAV manufacturing, assessing their throughput and process yield, and demonstrating robust clearance of model viruses like Adenovirus type 5 and Simian virus 40.

Adeno-associated virus (AAV) is highly inefficient at packaging its genome, with up to 90% of the formed AAV capsids being empty. The upstream cell lysis generates significant impurity burdens for downstream. A purification process, including harvest clarification, ultrafiltration/diafiltration, affinity chromatography for AAV capture, and anion exchange chromatography (AEX) for AAV polishing was developed for different serotypes. This talk will highlight:

• New chromatography and membrane filtration modalities that are used to overcome the manufacturing process challenges
• An increase in resin loading was found to enhance virus recovery from the affinity capture. 
• Over 40% increase in full capsids was achieved through AEX polishing.
  

This presentation will focus on the latest advancements in AAV polishing technology, crucial for enhancing the purity and potency of adeno-associated virus (AAV) vectors used in gene therapy. It will cover innovative methods for downstream processing, including chromatography and filtration techniques that effectively remove impurities and improve vector yield. The session will also discuss the impact of these technologies on the overall quality and scalability of AAV production.