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 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.

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.

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 innovative approaches to enhance impurity clearance using mixed-mode and mixed bed chromatography processes as well as anion exchange chromatography process in the presence of organic modifiers.

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.

Buffer requirements for large-scale purifications present significant facility storage and resource demands. Buffer concentrates, which minimize these constraints, have been successfully implemented in seven 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%.

Mammalian expression of recombinant proteins with the desired quality and post translational attributes can be extremely challenging due to low expression and poor purity. To address this challenge, we at Sanofi implemented a two-armed approach to optimize our expression and purification methods for difficult-to-express proteins. The Expi293F Inducible system combined with an alternative multipurpose affinity tag proved to be a great tool for mg-scale production of difficult-to-express proteins.

Accepting the new reality: modality diversity is the new normal
Identifying, selecting, implementing the right separation chemistries
Establishing work packages to drive platform implementation
Achieving harmonization across multiple locations
Developing new engineering approaches to address future modalities

Starting with a life-cycle strategy
Complimenting existing platforms/processes
Finding the balance between volume and capacity
Ensuring proper support infrastructure is in place
Everything has a cost - determining if/when intensification makes good sense

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.

In recombinant adeno-associated virus (rAAV) purification process, host cell proteins (HCPs) were reduced in multiple unit operations to ensure sufficient clearance in drug substance. Here, we demonstrated that additives could be introduced in affinity chromatography step to further reduce HCPs while maintaining rAAV genome recovery.

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.