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>Cell & Gene Therapy</span>
In situ hybridization (ISH) for localization of DNA/RNA hybrids in cytological preparations was first described in 1969 by Gall and Pardue. This method enables mRNA transcripts to be detected in tissue sections. Unlike expression analyses based on polymerase chain reactions, the exact localization of the target transcripts can be identified within the tissue…
An emerging application of viruses involves engineering them to treat diseases using a number of approaches. Broadly defined under the “virotherapy” umbrella, these include viral vectors used for gene therapy, oncolytic viruses, and viral immunotherapy. Although a majority of these products are in various stages of clinical development, the diversity of the therapeutic targets and wealth of future opportunities is encouraging. A significant challenge, as it is for any virus-based technology, is gaining a clear picture of the quality of a sample at any given point—from early research and development through manufacturing and product release. Of prime concern is the quantification of viruses, which in the past, has relied on slow, labor-intensive, subjective methods such as plaque titer assays and electron microscopic imaging. However, the diversity of new viral technologies now being used as the basis for innovative drugs and vaccines requires advanced, sophisticated analytical systems. In this white paper, we discuss how the real-time enumeration of viruses made possible by the ViroCyt® Virus Counter® 3100 can significantly enhance the pace of virotherapy product development…
Microbial contamination is of great concern in pharmaceutical and biotech manufacturing. Many organizations struggle with determining the root cause of contamination when it occurs, and identifying effective safeguards to prevent future contaminations. This article will introduce a new model for understanding microbial contamination in biopharmaceutical and sterile products and processes. The “Contamination Triangle” identifies the three factors necessary for microbial ingress into a sterile (or pure) system. The use of this model will provide guidance for contamination investigations, clarify the explanation of the contributing root causes in Non-Conformances, and assist in identifying risks and risk mitigation measures as part of a Failure Modes and Effects Analysis (FMEA) or other risk assessment method…
Implementation of “real-time” analytics (RTA) in processes for biologics is challenging from a technological and timeline perspective. Therefore, there need to be significant drivers from both a regulatory (quality) and a monetary standpoint to justify investment. Understanding how regulatory agencies define real-time analytics and the expectations for implementations (how and when) is a key component to rational decision-making, and dovetailing process improvement and facility design is important in the planning and development process…
Biologics Production Cell & Gene Therapy Cell Lines Mammalian Cell Culture Manufacturing Regulatory Viral Reference Materials Viral Vectors
at-line analytics biolgics design space biologics cpp critical process parameters design-of-experiments doe fda guidance fmea in-line analytics ind investigational new drug mabs monoclonal antibodies on-line analytics pat process analytical technology real-time analytics regulatory considerations rta
While playing an integral role in biotechnology and medicine, cryopreservation (CP) is often viewed as a “simple tool” and is overlooked as a critical and evolving component of cell and tissue bioprocessing. Despite this, cryopreservation serves as an enabling technology in numerous areas including the latest cell therapies. For example, over one third of the cells used in clinical trials are cryopreserved using the traditional methods, which in many cases yield suboptimal outcomes. Further, researchers still rely on the assessment of cell survival immediately post-thaw (within a few hours), and fail to account for the impact of cryopreservation-induced delayed-onset cell death (CIDOCD) which continues to impact survival from hours to days post-thaw.
Safety is typically viewed, perhaps unconsciously, as the result of a collection of factors, conditions, or behaviors. For example, consider “safety” in the context of personal, financial, or travel. With each, safety is defined as a set of component risks that have been managed to satisfactory levels for a particular situation. The same is true for product safety and risk, whether it be for raw materials or finished goods. The “safe” use of fetal bovine serum (FBS) is achieved by the management of controllable risks to a level that is acceptable for each particular application. For example, risk reduction requirements for research applications are not as stringent as for diagnostic, therapeutic, or manufacturing applications. Each end-user must decide on the level of risk reduction that is appropriate for their application…
For over 80 years, fetal bovine serum (FBS) and other animal-derived materials have been widely used in the production of vaccines, and more recently, biotherapeutics, for both human and animal applications. Ever since FBS was initially developed as a cell culture reagent, there have been efforts made to avoid the use of this critical commodity. The International Serum Industry Association (ISIA) recognizes the requirement for robust risk assessment and management, and has several ongoing programs designed to help mitigate the risk of using animal-derived materials. This article will provide an outline of the state of the industry and of these programs…
The world in general, and biopharmaceuticals in particular, are becoming increasingly complex and challenging. The ability to plan and execute a project to efficiently and effectively achieve a high-quality project goal is especially important for developing and manufacturing biopharmaceutical products. Managing projects ranging from product development, process characterization and validation, to building new manufacturing facilities requires straightforward, effective project management approaches and tools. But perhaps even more importantly, managing a project is a fundamental enabling skill required to manage both oneself as well as teams of people.
The proper handling of commonly used chemicals in bioprocessing is critical to maintaining a safe working environment as well as operational efficiency. Chemical mishandling can lead to failed batch processes, quality issues, as well as lost time and resources. As new technologies designed to help mitigate these safety risks become available, biomanufacturers have more opportunities to ensure that their production environments are safe. As a raw materials supplier, MilliporeSigma believes suppliers can play a critical role in terms of providing product and packaging solutions designed to minimize chemical handling risks…