In biopharmaceutical manufacturing, buffer preparation is traditionally performed manually by dissolving solid salts in water for injection (WFI) followed by offline mixing and specification testing. This method requires large buffer volumes, extensive infrastructure, and significant labor, often creating bottlenecks in production. To address these limitations and support process intensification, inline buffer preparation technologies have emerged…
BioProcessing Journal Posts
The number of cellular therapies in clinical trials and on the market has continued to rise significantly in recent years—and so does the need to maintain strict control over all manufacturing steps in order to reduce batch-to-batch variability. One potential source of product variability is the manual thawing of cryopreserved cells in a water bath, which can differ between operators. Additionally, water baths pose a significant contamination risk, making them less suitable for GMP environments. To overcome these challenges, several companies have developed water-free thawing devices that offer better control of the thawing process. However, these devices either accommodate only one vial at a time or lack U.S.
FDA 21 CFR 11 compliance in producing a computer-generated audit trail. Hence, we have developed a novel, water-free and dry-heat-based, fully programmable thawing device that is capable of thawing up to ten vials simultaneously and complies with 21 CFR11 requirements…
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 purpose of this study was to explore extremozyme production optimization. We found that maximum cellusases were obtained after 72 hours at 55 °C and 4.8 pH. Fructose was determined to be the optimum carbon source and peptone was the nitrogen source for cellulase production. Further, Tween 80 was used as a surfactant and ammonium sulphate served as a mediator to enhance the production of cellulases by A. fumigatus. The enzyme produced was subjected to purification by ammonium sulphate precipitation and gel filtration chromatography. The maximum concentration of cellulase was purified with 40% ammonium sulphate and gel filtration chromatography which resulted in a 3.5-fold purification. The purified enzymes were characterized with regard to optimum pH, temperature sensitivity, substrate specificity, and kinetic parameters such as Km and Vmax. Cellulase reached maximum activity at 55 °C and 5.5 pH. Metal ions like Ca2+, Mg2+, and Zn2+ had positive effects on cellulase activity. The Km and Vmax were found to be 4.34 µ M/mL and 7.29 mM respectively…
The current gold standard for the detection and identification of microorganisms has been in place for over a century. Petri culturing has been a solid diagnostic platform and its impressive run through history has served us well. However, modern requirements are challenging this traditional technology. Pressures within clinical and industrial applications have personnel demanding faster results while labs are forced to wait lengthy periods of time for confirmation of viable bacteria growth, or no growth. Depending on the organism, typical culturing results require anywhere from 24 hours to 21 days. It is time to give our culture-based friend a modern makeover to build on its outstanding reputation and enable it to compete in the modern world…
Risks in the pharmaceutical supply chain are not solely related to the active pharmaceutical ingredients (API). On average, excipients used in formulating medicines comprise 80% of each product and include a broad range of substances such as antiadherents, binders, coatings, disintegrants, diluents, flavors, colorings, preservatives, and sweeteners. Pharmaceutical excipients aid in the manufacturing process to protect, support, or enhance stability and bioavailability, and also make the product acceptable to the patient. They may also assist in product identification and augment the overall safety or function of the product during storage or use. Excipients represent an annual market value of approximately $4 billion worldwide, accounting for 0.5% of the total pharmaceutical market, according to industry experts…
The pitched-blade system is traditionally the choice for stirred tank bioreactors in large-scale production of animal cells in suspension culture. The packed-bed basket technology developed by New Brunswick Scientific provides a shear-free environment for large-scale (up to 100 L) production of animal cells. At present, little information is available on the utility of this system for the production of secreted proteins, especially in perfusion mode of operation. The perfusion process provides a homeostatic environment for optimal cell growth similar to that experienced by cells in vivo. In contrast, the batch culture approach does not appropriately model this homeostatic environment due to the depletion of nutrients and accumulation of waste products in the culture system. Thus, the objective of this study was to compare the growth and productivity of alkaline phosphatase (ALKP)-secreting Chinese hamster ovary (CHO) cells cultured in these two bioreactor types: pitched-blade bioreactors operated in batch mode versus packed-bed bioreactors operated in perfusion mode. CHO cells cultured in the packed-bed bioreactor, operated in perfusion mode, produced greater amounts of ALKP compared to cells cultured in the pitched-blade system run in batch mode. These observations suggest that continuous exposure of cells to fresh culture media and the shear-free culture environment provided by the Fibra-Cel growth matrix disks offered more favorable growth conditions for CHO cells, allowing for either greater cell proliferation (higher density) or greater protein production on a per-cell basis. Overall, the results of this comparison study suggest that packed-bed bioreactors provide significant advantages for moderate-scale production of cells. The benefits of this bioreactor system may translate to large-scale cell culture for generating secreted protein products useful in medical applications…
The Leahy-Smith America Invents Act (AIA) and the Biologics Price Competition and Innovation Act (BPCIA) are dramatically reshaping business strategies designed to protect intellectual property and regulatory exclusivity rights granted by the United States Patent and Trademark Office (PTO) and the Federal Drug Administration (FDA) to the sponsors of new and follow-on versions of many biopharmaceutical products. The AIA alters many provisions in the U.S. patent statutes, most notably long-standing policies and practices relating to the conditions for patentability of an invention, particularly 35 USC §§ 102 and 103 relating to novelty and non-obviousness, respectively, and introduces new provisions relating to post-grant review proceedings and prioritized examination that will affect the business plans of academic and corporate institutions seeking to discover and develop health care products that serve unmet medical needs or provide consumers with safe sources of drug products at a low cost. The BPCIA implements many new provisions relating to the abbreviated review and approval of follow-on biopharmaceutical products. Important aspects of the BPCIA, particularly issues identified in draft guidance documents that were released by the FDA in February 2012 concerning the characterization, comparison, and evaluation of reference and follow-on macromolecules, will be discussed in this article. Political issues that may jeopardize the BPCIA, economic considerations faced by drug product sponsors, and public reaction to the guidance documents are also discussed in this final article of a three-part series describing key provisions of the AIA and the BPCIA that affect intellectual property and regulatory exclusivity rights of institutions having an interest in developing novel or follow-on versions of biopharmaceutical drug products…
BacMam transduction technology combines the ability of baculoviruses to maintain and amplify large gene inserts with the desirable post-translational characteristics of proteins expressed in mammalian cell lines. Improved versions of BacMam expression vectors demonstrate increased transduction efficiencies and wider ranges of permissible, mammalian host cells. BacMam technology has been used successfully in the fields of cell-based assays for drug discovery and for imaging of subcellular structures and functions. The development of a large-scale platform for the transient expression of recombinant proteins using BacMam transduction would significantly improve the current transient transfection methodology. We report our initial results obtained using BacMam constructs expressing a humanized rIgG. Serum-free suspension cultures of HEK-293 and CHO-S cells were cotransduced with BacMam viruses at total multiplicity of infection ratios between 10 and 20. Purified yields of 18.9 mg/L were obtained for shake-flask cultures of HEK-293 cells under nonoptimized conditions. These results illustrate the potential of the BacMam system to produce significant quantities of recombinant proteins in mammalian host cells…
Advances in industry and medicine have led to the engineering of complex “designer” proteins, such as antibodies in targeted therapeutics and enzymes in process development. The ability to easily generate an almost infinite number of variants at the DNA level has increased the demand for improved protein expression methodologies to fully capture what can be produced genetically. Often, the protein of interest is eukaryotic in origin and may require posttranslational modifications specific to its native host or may be toxic to the host cells expressing them. Cell-free protein expression systems have allowed us to step beyond the limits of traditional in vivo expression methodologies by decoupling protein expression from host cell viability. Furthermore, the ability to produce complex proteins using cell-free transcription/translation systems uniquely enables high-throughput directed evolution and protein engineering efforts. Several cell-free protein expression systems have been developed in the last decade with recent advances focusing on special folding or assembly environments. Equally as important is the capability to transition from the in vitro system to larger-scale in vivo expression, while maintaining activity of the target protein…