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Rapid Antigen Production in Pseudomonas fluorescens Utilizing a High-throughput, Parallel Processing Approach

Pseudomonas fluorescens, a Gram-negative bacterium, has been specifically developed as a protein production platform to enable rapid identification of strains capable of expressing high titers of soluble and active protein. Pfēnex Expression Technology (TM) employs a toolbox of engineered P. fluorescens host strains, including protease deficient mutants and chaperone/ foldase overexpressors combined with a set of expression vectors encoding a variety of transcription and translation regulators, as well as a collection of periplasmic secretion signals. High-throughput automated transformation, growth, and…

Expansion of T-cells using the Xuri Cell Expansion System W25 and WAVE Bioreactor 2/10 System

Immunotherapeutics include drugs and biologics that render therapeutic benefit by harnessing the power of the immune system. The promise of immune-mediated therapies is to target specificity with a consequent reduction in off-therapeutic effects. Immunotherapeutic products can be classified broadly into (1) active immunotherapy (therapeutic vaccines), (2) adoptive cellular immunotherapy (transfer of immune cells, genetically modified T-cells or precursor cells) or (3) passive immunotherapy (antibody or receptor ligand administration). Recent scientific advances have led to clinical trials of both active and…

Microbial Cultivation In Different Scales in the CELL-tainer® Rocking Single-use Bioreactor

Single-use bioreactors are applied in the biopharmaceutical industry for mammalian cell culture processes. The low gas-liquid mass transfer coefficient generally achieved in these systems does not allow for the compatable application of microbial processes in these bioreactor systems. The CELL-tainer® technology combines a vertical and horizontal movement in a rocking bioreactor concept, so that the turbulence and power input is intensified. In this system, mass transfer rates comparable to stirred tank reactors are achievable. Thus, volumetric oxygen transfer rates (kLa)…

The Importance of Thresholding in Imaging Analysis of Protein Aggregates

Dynamic imaging particle analysis (DIPA) shows much greater sensitivity to transparent particles, such as protein aggregates, than light obscuration can. While not yet fully accepted in industry compendia, DIPA is being used increasingly in the formulation process for characterization of sub-visible particulates in biologics. DIPA measures particle size and shape by first creating a binary image based upon a defined threshold from the background value for each pixel in the image. The resulting binary image is used for all particle…

Anticipating Success: Meeting the Inherent Challenges of Complex Drug Substances

Realizing the full potential of a novel injectable drug compound is no small task. In the months and years that lead from the exciting discovery phase to the rigorous demands of a commercial launch, unexpected scientific and technical challenges can slow development to a halt, often at key stages. A careful, systematic approach to identifying where and why these roadblocks can occur is fundamental to staying on course. Just as important is a robust, repeatable process design focused on retaining…

Designing Quality into the Product: Early Developability Assessment in Biopharmaceutical Development

The ultimate objective in the development of any new therapeutic candidate is the validation of its mechanism of action and therapeutic efficacy in a clinical setting. Three important areas of product development critical to determining the success of a new drug candidate are: manufacturing, safety, and delivery of the product to patients. Early attrition observed during preclinical stages can often extend development timelines or require additional process optimization, therefore costing the developer more time and money. It is desirable to…

Validation of the New Single-Use Freeze-Pak™STS Storage and Transport Solution Containers

Production of biotherapeutics whether for clinical development or large scale manufacturing campaigns intended to be converted to final drug product often involves frozen storage. Frozen storage provides manufacturing process flexibility while enabling long-term product stability. Products are frozen and stored using a variety of technologies including stainless steel vessels, bottles, carboys and single-use bags. Use of bags has become popular due to their low investment cost and process flexibility attributes. Single-use bags intended for freezing and storage are often made with films using EVA and/or LDPE with product routinely blast frozen and stored to -30°C in a cold storage warehouse. During freezing and transport, the bags will typically experience temperatures well below -30°C ranging from -50 to -80°C. Under these conditions, the bags have to endure a wide variety of stresses (film brittleness, volume expansion, etc.) impacting integrity. With applications (like working cell banks for example) requiring lower temperatures for maintenance of long-term product stability, frozen storage films and containers designed for these conditions are needed. The new single-use Freeze-Pak™STS (FP-STS) frozen storage and transport solution containers from Charter Medical, Ltd. are manufactured using a unique polyolefin monolayer film designed for freezing applications The FP-STS bags (including tubing and connectors) have been validated for storage to -80°C, while the Freeze-Pak™ film remains flexible to temperatures as low as -196°C. The new Freeze-Pak™ STS bags deliver the flexibility and durability required for frozen storage and transport.

E. coli Cultivation in a 12L and 120L CELL-tainer™ Single-Use Bioreactor

Single-use bioreactors are usually applied in the biopharmaceutical industry for mammalian cell culture processes. For microbial processes, concepts like the CELL-tainer® technology allow comparable gas-liquid mass transfer rates like in stirred tank reactors. In the CELL-tainer, the rocking motion of the bag is generated with a combination of a vertical and horizontal movement. Due to a 2-D rocking motion, the turbulence in the liquid is intensified. Thus, volumetric oxygen transfer rates (kLa) of over 400 h-1 could be achieved. Recently, the successful scale-up of an Escherichia coli nutrient-limited fed-batch cultivation from the 15 L to the 150 L scale in the CELL-tainer single-use bioreactor has been conducted. A final biomass concentration of 45 gL-1 within 24 hrs was obtained in cultivations proving the general suitability of this reactor concept for the application of bacterial processes. The combination of intelligent software sensor control strategies and currently improving (single-use) sensors will lead to a reduction of current drawbacks and improve control of bacterial fed-batch processes. The availability of single-use bioreactors for microbial cultivations widens their potential, not only in biopharmaceutical processing, but also as a pre-culture bioreactor for large scale processes and as a suitable tool in bioprocess development.

Driving Optimal Cell-Level “Observability” and Data-Driven Process “Guidance”

In the spirit of implementing PAT (Process Analytical Technology), biopharmaceutical companies are striving to gain a more fundamental understanding of what is happening to the cells within their bioreactors. Implementation of online tools like Raman and Dielectric Spectroscopy are helping to provide insight, yet there is still a gap integrating this data with off-line measurements such as cell density, viability, metabolite levels and titer. Bend Research, in collaboration with major biopharmaceutical companies, is working to advance MAST, a modular, automated sampling platform that can provide samples directly from bioreactors to analytical devices, while maintaining process sterility. In this webcast, Clint Pepper, Ph.D. and Lisa Graham Ph.D., P.E. of Bend Research focus on this overall cell-level “observability” concept and illustrate how the MAST platform is incorporated.

Selecting a Transfection Reagent for Large Scale Protein Production in Suspension 293 Cell Types

Decrease time to produce usable protein by maximizing target protein yields through transient transfection. The TransIT-PRO® Transfection Kit uses animal origin free components designed for high and reproducible nucleic acid delivery into suspension CHO and 293 derived cells. Since it is compatible with varied media formulations, the same media can be used for both transient and stable expression. The TransIT-PRO outperforms linear PEI in protein yield, while providing a cost-effective alternative to FreeStyle™ MAX and 293Fectin™ Transfection Reagents.