Development of a chemically defined, animal-component-free ex vivo expansion process for activated human T cells

T-cell based immunotherapy applications have recently drawn great interest and notoriety due to their clinical potential as next-generation life-saving therapies for cancer patients. Generation of sufficient, desired T-cell populations is an essential task for the successful development of T-cell based immunotherapy, requiring an effective, scalable, and consistent ex vivo manufacturing process. A suitable chemically-defined (CD), animal-component-free (ACF) T-cell basal medium would significantly foster the establishment of such a process. Presented in this study is the development of a CD, ACF T-cell basal culture medium that is scalable and suitable for the manufacture of adoptive T-cell therapy products. A ‘quality of design’ approach coupled with spent media analysis was utilized to examine the effects of various key media compositions such as amino acids, vitamins, minerals, and lipids on activated human peripheral blood-derived T cells proliferation. The resulting CD, ACF basal expansion medium was comparable or superior to media containing serum (RPMI + 10% FBS) or serum-derived human albumin in the culture of both CD4+ and CD8+ T-cell populations. Further supplementation with proper cytokine combinations verified and demonstrated the CD, ACF basal media’s performance in effectively supporting the derivation of major T helper subsets, such as Th1, Th2 and Treg, from naïve CD4+ T cells. In addition to its utility in static culture conditions using T-flasks, the CD, ACF basal medium was subsequently evaluated for its potential application in other commonly used expansion vehicles including spinner flasks, culture bags, and G-Rex plate systems. A total expansion of 50 and 80-fold was achieved through the G-Rex plate and culture bag systems, respectively, when cultures were maintained for up to 14 days accompanied by a single media replenishment event suggesting the potential for even higher yields with further optimization and feeding steps. In all cases, the CD, ACF medium delivered the best growth profile while maintaining high viability and desired T-cell phenotypes (e.g. % of CD62L+ cells). The evaluation results demonstrate the improved performance delivered by the CD, ACF medium over serum-containing media and its suitability for use in the manufacture of adoptive T-cell therapy products

Low-cost cell-based production platform for seasonal and pandemic influenza vaccines

Influenza-related illnesses have caused an estimated over million cases of severe illness, and it has about hundred thousands of deaths worldwide annually. Traditionally these vaccines are produced in embryonated chicken eggs. However, in the case of a pandemic outbreak, this egg-based production system may not be quickly enough to meet the surging demand. The efficacy associated with egg-based vaccines are low in recently years. The raising concerns with egg-derived vaccines is resulting in the spurred exploration of alternatives. MDCK cells are becoming as an alternative host to embryonated eggs for influenza virus propagation. Although MDCK cells were considered to be a suitable host for the virus production, their inability to grow in suspension still limits the process of scale-up and their production capability. Please click Additional Files below to see the full abstract

Development of an animal-component free insect medium for the Baculovirus Expression Vector System (BEVS)

Insect cells derived from Spodoptera frugiperda have been widely used with the baculovirus expression vector system (BEVS) for the production of recombinant proteins and adeno-associated viruses (AAVs) due to their ease of culture, scalability in high cell density suspension cultures, and high protein expression levels. Traditionally, insect cells are cultured in an undefined medium containing yeast hydrolysate and cod liver oil, however, there is an increasing push to use chemically defined, animal-component free medium to minimize any potential contaminants and decrease lot-to-lot variability while maintaining high cell growth and production. In this case study, an animal-component free insect medium was developed utilizing Rational Culture Media DesignTM and evaluated with Sf9 cells. Using a traditional formulation as a starting point, the final medium was developed by optimizing multiple nutrient groups in the basal medium, replacing the animal-derived components, and screening several yeast hydrolysate sources. By utilizing multifactor design of experiment software, various nutrient groups were screened including amino acids, vitamins, and metals. The metals group was identified to have the most impact on cell growth and productivity, and therefore concentrations of metal components were further optimized. In addition, the animal-derived components in the starting formulation, cod liver oil and cholesterol, were replaced with animal-component free fatty acids and synthetic cholesterol, respectively. The concentrations of these components were optimized to achieve better growth performance and production while also sustaining formulation stability and streamlining manufacturing processes. Finally, yeast hydrolysate is a well-known, undefined component that is crucial for insect cell growth and productivity. To minimize lot-to-lot variability, the yeast hydrolysate concentration was significantly lowered, and multiple yeast hydrolysate sources and lots were evaluated to determine the highest quality source. As a result, an animal-component free insect medium was developed that had improved growth performance and comparable productivity to a widely used commercially available animal-derived medium

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead