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Check out our list of publications below.

Title: Characterization of Oxygen Mass Transfer Rate (kLa) Using a Statistical Design of Experiment (DoE) for Bioreactor Scale-Up.

Abstract: Bioreactor production development and optimization are typically performed at a small scale to reduce their cost and time requirement. Bioreactor production is scaled-up often based on keeping volumetric mass transfer coefficient (kLa) constant. To facilitate a successful transfer of an optimized small-scale cell culture bioreactor production to lab, pilot, and industrial scale, the kLa profiles for these bioreactors should be determined. Although a number of correlations and predictive models for kLa are available, they do not have universal applicability. The purpose of this study was to use a statistical Design of Experiments (DoE) approach to characterize kLa in 3L, 7L, and 50L bioreactors, and hence to use this information for bioreactor scale-up. Dissolved oxygen sensors were used to measure dissolved oxygen concentrations in the bioreactors. kLa was determined by using the dynamic reoxygenation method. Response surface design of experiment was used to characterize kLa in the bioreactors. Agitation, air flow rate, and volume were included in the experimental design as input parameters, and kLa was used as the output parameter. Results of these studies is discussed in this presentation. The correlations determined here will be used as a tool for bioreactor production development, scale up and tech transfer.

Title: Characterization of Oxygen Mass Transfer Rate (kLa) Using a Statistical Design of Experiment (DoE) for Bioreactor Scale-Up

Abstract: Bioreactor production development and optimization are typically performed at a small scale to reduce their cost and time requirement. Bioreactor production is scaled-up often based on keeping volumetric mass transfer coefficient (kLa) constant. To facilitate a successful transfer of an optimized small-scale cell culture bioreactor production to lab, pilot, and industrial scale, the kLa profiles for these bioreactors should be determined. Although a number of correlations and predictive models for kLa are available, they do not have universal applicability. The purpose of this study was to use a statistical Design of Experiments (DoE) approach to characterize kLa in 3L, 7L, and 50L bioreactors, and hence to use this information for bioreactor scale-up. Dissolved oxygen sensors were used to measure dissolved oxygen concentrations in the bioreactors. kLa was determined by using the dynamic reoxygenation method. Response surface design of experiment was used to characterize kLa in the bioreactors. Agitation, air flow rate, and volume were included in the experimental design as input parameters, and kLa was used as the output parameter. Results of these studies is discussed in this presentation. The correlations determined here will be used as a tool for bioreactor production development, scale up and tech transfer

Title: Strategies to Accelerate Process Development from Preclinical to Manufacturing for Gene Therapy

Abstract: Gene therapy is a fast-growing industry. Demand on its products such as pDNA, AAV, retrovirus, lentivirus, etc. has significantly increased during last 10 years while its production challenges remain unresolved. It is a complex process with a number of unoptimized and low yielding steps. A typical process development (PD) cycle from preclinical to late phase manufacturing (Mfg) takes 3 to 5 years. It is desired to rapidly advance to manufacturing for early clinical candidates using well established and high yielding processes. The following strategies were successfully employed by BIB to accelerate the process development activities: (1) A platform approach for early stage process development, (2) Efficient DoE application for process optimization and characterization, (3) Interaction improvement between PD and Mfg to streamline scale-up, tech transfer and manufacture support, (4) Development of a new PAT tools application to understand process design space and hence develop a control strategy. Case studies for BIB AAV and pDNA production platforms will be covered in this presentation.

Title: Purification Process Development of Plasmid DNA for Gene Therapy

Abstract: Plasmids are small double stranded DNA molecules that are separate from the chromosome. Plasmids are present naturally in bacterial cells and are commonly used as raw materials in mRNA and gene therapy vaccines. These plasmids can be used as the gene template for mRNA vaccines, or to provide the necessary genes, such as rep/cap/gene of interest, in adeno-associated virus (AAV) processes. The current work outlines MilliporeSigma’s collaboration with Boston Institute of Biotechnology, to develop unit operations for the purification of a plasmid DNA used for AAV gene therapy application. Currently the purification of pDNA in a pure form ready for therapeutic use still presents various challenges starting from the lysis step. It also requires development of highly reproducible and scalable processing methods that meet regulatory manufacturing standards. The development here focuses on purification of the supercoiled isoform of pDNA and the use of scalable technologies on process steps of lysate clarification, ultrafiltration/diafiltration, capture using membrane ion exchange chromatography, and sterile filtration

Title: Strategies to Accelerate Process Development from Preclinical to Manufacturing for Gene Therapy

Abstract: Applications of adeno-associated virus (AAV) in gene therapy has increased significantly in the last two decades due to its remarkable safety profiles and efficiency of gene delivery into various tissues. Demand on recombinant AAV vectors has also increased to fulfill pre-clinical and clinical requirements. Helpervirus-free mediated transient transfection has been widely adopted. Low titer and process scalability remain two key issues in AAV manufacturing. The productivity of AAV vectors is often affected by the AAV serotype, cell culture type, the size of Gene of Interest (GOI), cell density, transfection method, plasmid DNA concentration and other factors. Based on the risk assessment of these factors that may impact AAV production with helper-virus-free mediated transient transfection, we have evaluated 6 process parameters using JMP’s Definitive Screening Design. In this study, critical process parameters (CPP) were identified as plasmid DNA to cell density ratio, cell density per surface area and incubation time post transfection. Their operating ranges were established for optimum and robust AAV production, which can be applied while scaling up to manufacturing. AAV expression level was improved to 2E11 vg/mL.