Allogeneic iPSC-derived iNK and iT cell therapies have shown encouraging preclinical and clinical promise.  These therapies have the potential to treat a wide range of indications and provide a limitless source of readily available doses to numerous patients. As therapeutic technologies progress, the requirement for the development to adequately support clinical-to-commercial scale production becomes more critical. To that end, this presentation will highlight the downstream processing, purification, and drug product formulation strategies that meet the requirements of both patients and cell therapy manufacturers.

This paper will detail elements of an investigation into the formation of particles in a drug product formulation. The paper will focus on the drug substance components of the investigation.

This laboratory has developed a novel family of polymer fiber stationary phases to affect the isolation and purification of exosomes using a hydrophobic interaction chromatography elution scheme.  Here we will demonstrate the breadth of applicability of the approach, extending it to further sources of exosomes including bovine milk and plants. The same method has now been applied to lentiviruses and adeno-associated viruses (AAVs) and lipid nanoparticle (LNP) vectors as well. The ability to affect separations across this group of vector species, using the same process platform and protocol is seen as a tremendous advantage on the research and clinical scales.

This presentation will cover the following areas:

• AAV purification historry: density gradient centrifugation to chromatography
• Current practices: process design, efficiency, capability, scalability, consistency, yield to cost reduction
• Challenges & solutions: process- and product-related impurity removal such as empty capsid and aggregates

Filtration is an essential component in biologics downstream manufacturing. The COVID pandemic has caused a global supply shortage in filters used in various downstream processing steps. To ensure manufacturing continuity and uninterrupted delivery to patients, innovative solutions are needed to overcome these challenges. Alternative filter evaluations and sizing studies have been performed to mitigate stockout risks for several filters used in commercial biologics manufacturing.

This study details development and provides insight on the alternative approach to using mixed-mode CHT chromatography with complex biologics within the downstream purification process.

Antisense oligonucleotides are short single strand modified RNA/DNA sequences that are designed to treat genetic disorders by eliminating target mRNAs via specific binding. The safety and efficacy of these therapeutics can be affected by their purities, and therefore, a robust purification process is required. In this study, we explored novel approaches to enhance impurity clearance using anion exchange chromatography polishing step using dual salt systems and organic modifiers.

Protein separation is an important purification step in a biopharmaceutical downstream process. Prior to column packing, homogenous resin slurry is necessity for optimal column packing and effective chromatographic purification. Agitation speed, packing buffer, and resin solid percentage are key factors to achieve the homogeneity. In this study, computational fluid dynamics (CFD) is used as a tool to guide those operating parameters for slurry suspension in a stirred tank and thus providing a more consistent column packing process. The result of this work can be applied to predict optimal slurry tank parameters for other types of resin.

3D design and printing bioprocessing structures enables the chemical and physical characteristics of downstream media to be specifically tailored to the product of interest, particularly important due to the emerging popularity of advanced modalities. In this study we apply high resolution imaging to view multiple length scales analogous to packed bed chromatography that informs the design of next-generation DSP materials.

Clearance of endogenous and adventitious viruses is an important consideration for any mammalian cell-derived biotechnology product. Process changes in the investigational phase may impact clearance capabilities depending on the applied unit operations and parameters. Data from an in-house database created to evaluate these process parameters and their impact on clearance will be presented.

This talk focuses on designing a process relevant impurity clearance challenge studies. We used risk-based approach to identify process-related impurities that warranted impurity clearance challenge studies, and applied three different strategies to design the studies: upstream worst case material, column overloading approach and by-pass approach. Data generated will be used to set acceptance criteria wihtin the process control strategy.

In this investigation, we employed AEX chromatography to simultaneously enrich full AAV capsid and eliminate endotoxin from the final drug product. By optimizing the column conditions, we achieved a high level of AAV capsid purity, as well as a significant reduction in endotoxin levels. This strategy offers a promising solution for the production of safe and effective AAV-based therapeutics.

Manufacturing strategies and progress towards platform processes for adeno-associated virus (AAV) production have seen substantial advancement in support of the impressive clinical success for this modality. This presentation will provide a case study for the development of a robust, high yield downstream process for the production of AAV serotype 5. Key findings will be discussed in addition to pitfalls and challenges in this development work.

High throughput purification development has become an integral part of purification development by providing an order of magnitude reduction in material and time requirement. It can be especially valuable since material limitations are often a challenge for AAVs. An initial screening of several process operating parameters using small amounts of material can be performed to narrow down the development space to focus on and further optimize the process. Here we discuss the various high throughput approaches utilized in Sanofi for developing several downstream unit operations including clarification, affinity chromatography, polishing AEX chromatography, etc.

Recovery out of harvest is the least understood step in downstream purification of AAV vectors. Complexity of lysed cell culture coupled to relatively low protein concentration of AAV product makes it very difficult to analytically investigate this process space. Here we evaluate the ability of dynamic light scattering (DLS) to serve as analytical characterization tool that would allow to investigate AAV capsid aggregation in a complex matrix of clarified harvest.

This talk may cover topics including selection of appropriate purification methods, process optimization, and quality control measures to ensure high-quality plasmid DNA for gene therapy applications.