Lentiviral vectors (LVV) are widely used in an increasing number of approved cell and gene therapies, and benchmarking is important in evaluating production processes and products. We have produced a lentiviral vector reference material (LVV-RM) that expresses the common marker gene (green fluorescent protein [GFP]) to enable data comparisons and support LVV research programs. To generate that reference material, we have applied our previously developed fed-batch process to a 100 L production using an inducible LVV-producer cell line. The LVV material was harvested three days post-induction from a 200 L single-use bioreactor. A downstream purification process was also developed and scaled-up to meet production requirements: It consisted of nuclease digestion, clarification by depth filtration, chromatography capture using CIM-QA monolith anion exchangers, and ultrafiltration/diafiltration using a hollow fiber membrane prior to bulk-filling the final product. A total of 4.4 L of diafiltered and concentrated LVV product was obtained (9.2 E10 particles/mL, 3.6 E7 TU/mL) and stored at -80°C. Nearly 8000 vials are now available to the LVV community via the American Type Culture Collection (ATCC).
Category: <span>Biologics Production</span>
Efficient bioprocess characterization is essential for both regulatory compliance and commercial viability of biologics. Traditional approaches using resolution III/IV screening designs followed by response surface methodology are time-consuming, costly, and not always effective in identifying the important experimental effects. Definitive screening designs (DSDs) represent a novel class of three-level screening designs that can simultaneously evaluate main effects and quadratic relationships. While DSDs are increasingly used in bioprocess development, practical implementation guidelines remain limited. This case study bridges this gap by introducing a model-based framework to identify critical process parameters (CPPs) and optimize operating ranges for robust biologics production using plasmid DNA (pDNA). Minimal 14-run DSDs evaluated six input parameters and successfully identified CPPs and optimal operating ranges. This approach reduces experimental requirement by >50% compared to traditional designs, providing an efficient and economical strategy for bioprocess characterization and optimization.
This study assessed a novel statistical approach using space-filling designs (SFDs) and self-validating ensemble modeling (SVEM) machine learning to efficiently identify key process factors using recombinant adeno-associated virus type 9 (rAAV9) gene therapy manufacturing as a case study. Based on risk assessment of parameters that may impact rAAV9 production, we have evaluated six process parameters using 24-run SFDs generated by the JMP statistical software. SFDs are a new class of design of experiment (DoE) created with the objective of covering the entire design space as completely as possible; this in turn allows more accurate modeling of complex response surface behavior typically found in bioprocesses.
The price per patient for protein-based and monoclonal antibody (mAb) therapies runs into thousands of dollars per patient each year. These therapies cost considerably more to manufacture than small molecules. Hence, if mammalian or insect cell lines expressing high protein titres can be selected and optimized for protein expression using microscale bioreactor models early in development, then manufacturing costs can be reduced significantly…
For the ongoing 2014 Ebola virus outbreak, all viable options and technologies need to be evaluated as potential countermeasures to address this emerging biological threat. Novavax, Inc. has a rapid, practical vaccine development and manufacturing platform with the capability to deliver clinical trial material and, ultimately, commercial doses in response to novel infectious disease agents. This report describes the application of our platform technology for the successful generation, manufacture, and release of a clinical batch of Zaire ebolavirus glycoprotein nanoparticle vaccine three months from project initiation…
This paper describes how a biopharmaceutical product development effort can be structured to identify, understand, and plan activities and goals required to efficiently and rapidly deliver new products and therapies to patients. Although the paper focuses on manufacturing, the approach can be used for all aspects of pharmaceutical product development from establishing an intellectual property position, developing a comprehensive manufacturing plan, to creating a marketing program…
The purification of PHB-01 plantibody derived from tobacco leaves imposed difficulties when the plantibody solid-liquid extraction design was performed. Thus, our study focused on assessing a combination of an aqueous two-phase extraction (ATPE) procedure and affinity chromatography for solving some of the issues in plantibody purification. This was done using a complete factorial redesign, different polyethylene glycol (PEG)/K2PO4 proportions, and pH values in each partitioning variant.
Sea lice are the most problematic marine pathogens the salmon industry has to deal with, significantly affecting Europe and America. The worst offenders are genera: Pseudocaligus, Caligus, and Lepeophtheirus. Over €305 million in losses are estimated. Recent results have suggested that subolesin/akirin/myosin32 are good candidate antigens for the control of arthropod infestations such as sea lice. The aim of this study was to design and optimize the purification step of MY32/Ls protein to obtain the active pharmaceutical ingredient against sea lice. Non-chromatographic purification strategies were employed, based on published works, to establish rupture, washing, solubilization, and refolding conditions…
Stem cell-based regenerative medicine has great potential to advance the therapeutic treatment of human diseases. Among the various stem cell platforms, mesenchymal stem cells (MSCs) represent one of the most promising options. Currently, there are over 500 clinical trials based on MSCs registered at the NIH’s ClinicalTrials.gov website. Although successful expansion of MSCs in vitro has been well-established, higher-yield, billion-cell expansion of MSCs remains a bottleneck. In this study, we successfully demonstrated large-scale culture of human adipose-derived mesenchymal stem cells (AdMSCs) in an industrial, single-use vessel at 3.75 L scale.