Perfusion systems for animal cell cultures are increasingly used for high cell density processes to enhance the productivity of bioreactors. There are four different process modes that can be used in animal cell cultivation: batch, fed batch, continuous culture, and perfusion culture. In both batch and fed batch, the metabolite concentrations cannot be kept constant and the accumulation of catabolites like lactate and ammonium limits the process duration to about ten to 15 days. In contrast, with continuous and perfusion processes, there is a constant influx of fresh cultivation medium and a corresponding removal of recombinant protein and catabolites. The concentrations of metabolites remain relatively constant and the process duration is not limited by the buildup of waste products. This extends the duration of these cultures to several weeks or months. The resulting steady-state conditions for metabolites can enhance cell-specific productivity and product quality, for example, by improved glycosylation or reduced aggregate formation. In continuous culture, cells are removed in the effluent and this limits the product output per liter of bioreactor volume (volumetric productivity). In contrast, substantially higher cell concentrations are attained in a perfusion system because the cells are retained in the bioreactor resulting in increased volumetric productivity. A five- to 20-fold improvement over batch cultivation has been reported for perfusion cultures…
Category: <span>Biologics Production</span>
Continuous cell lines of Drosophila have now been in use for more than two decades as hosts for the expression of heterologous gene products. The most popular are the Schneider lines 2 and 3 (S2, S3) and a few derivatives of the Kc line. These have been widely employed for both stable and transient gene expression. The S2, in particular, is an exceptionally versatile system that has proven to be useful for high-level protein production. S2 cells have been used for the expression and analysis of intracellular, secreted, and membrane-associated proteins. This includes cytokines, antibodies, receptors, and viral antigens. These have all been shown to be authentically-processed, biologically active, and produced to high levels. The cell line is ideally suited for the development and industrial-scale manufacturing of a wide range of biopharmaceuticals including vaccines and therapeutic protein products. Compared to conventional technologies, the strengths of the S2 expression technology lie in its quick access to proteins, excellent protein expression capability, scalability, regulatory-friendliness, and applicability to high cell density perfusion cultivation…
Single-use systems (SUS) have been employed in biopharmaceutical manufacturing for over 15 years. Consistent year-to-year growth has been seen both in the total volume of sales and the number of manufacturing steps in which SUS are being used. It is projected that the majority of commercial-scale biologics manufacturing will be done in disposable equipment in the near future because of improvements in SUS design and innovation. These devices can significantly reduce capital costs (for example, stainless steel vessels), eliminate equipment cleaning and sterilization, improve turnaround times, and reduce concerns about microbial contamination within a production facility. As reported in this year’s 8th Annual Survey of Biopharmaceutical Manufacturing, however, the rate of growth in SUS use may be slowing. Contributing factors are: 1) the economy; and 2) the need for more consistent product standardization (e.g., design, quality, and leachables/extractables [L&E]) data that regulators and industry end-users can accept with confidence…
Single-use processing solutions spanning both upstream and downstream applications are being embraced by the biopharmaceutical industry. The advantages of the single-use approach to industrial cell culture versus traditional stainless steel and/or glass bioreactors has resulted in the recent commercialization of several single-use bioreactors at small (3–15L), intermediate (50–500L) and large scales (>1000L). Recent innovations are combining the features of conventional bioreactors with the ease-of-use benefits associated with single-use technology for the optimization of mammalian cell growth and recombinant protein expression. This article will provide a detailed characterization of the single-use Mobius® CellReady 3L Bioreactor capabilities as compared to a glass bioreactor, in terms of CHO cell growth, mixing, and volumetric mass transfer coefficients (kLa) for oxygen…
While global economies continue to struggle back from the recent recession, we are seeing clear evidence that budgets in most areas of biopharmaceutical manufacturing are returning to pre-crisis conditions. Because of the essential nature of pharmaceutical products and health services, healthcare sectors tend to be more recession resistant and return to growth sooner than other industries. In fact, respondents to our 8th Annual Report and Survey of Biopharmaceutical Manufacturing projected only increases for all budget areas this year. This is a change from last year where budgets continued to show decreases in areas ranging from outsourcing production, hiring new scientific staff, and new facility construction. As the financing crunch lightens, biomanufacturing facilities are reopening their wallets. However, anecdotally, vendors to the industry are indicating that buyers are continuing to be much more cautious with their spending. Sales cycles continue to be drawn out, and many end users are demanding more milestone performance and risk sharing from suppliers…
Many biological systems and chemical reactions are inherently interactive, and the polymerase chain reaction (PCR) is a notable example. The qualities of the components in PCR explain this interactivity. Free nucleotides, template DNA input, and PCR product are charge-attracted to the magnesium ions required for a functional polymerase. Whereas overall salt content in the reaction can serve to neutralize the influence of the nucleic acid charge, too much or too little salt interferes with the processivity of the polymerase. In addition, secondary and tertiary structures, and guanine-cytosine (G-C) content in the template DNA and PCR product can interfere with the ability of primers to bind to the correct target. In such an instance, a commonplace approach is to add disruptive agents such as DMSO to the reaction that also interferes with the activity of the DNA polymerase. Due to these complex interactions, many researchers resort to simplistic optimization schemes such as varying free magnesium concentration or altering the annealing temperature in the cycling protocol. If the simple approach fails, a common “solution” is to search for alternative target sites and start over with a new set of conditions. This approach may not suit all investigators and may not be possible for certain reactions. With difficult PCR reactions, a lack of robustness may result in a failure to subsequently repeat findings if the operator, the equipment, or the laboratory changes. Here we demonstrate the use of a statistical manufacturing method to make PCR optimization more robust. Based on the statistics developed by Taguchi, we show reliable PCR amplification with greater sensitivity than previously published by others using primers and a target gene in the G-C rich herpes simplex virus type 1 (HSV-1) genome. When applying the same statistical method to a non-interactive enzymatic reaction, there was no indication for the need to change the reaction components…
The propagation of the yeast Saccharomyces cerevisiae was optimized using a Taguchi parameter design (TPD) L9(3 4) to produce bioethanol from an amylaceous material. The response factor selected was the specific growth rate of the yeast as calculated from the slope of the linear portion of its growth curve (neperian log cell concentration versus time). The reason is that the greater this rate, the higher the number of viable cells in the fermentation broth capable of ethanol production. The control factors selected were the initial amount of inoculum in the medium, the amount of glucose, the temperature, and the shaking speed which are the chemical and physical variables that most affect the growth behavior of this yeast. The noise factor selected was the initial peptone concentration in the medium. Statistical analysis and factorial split-plots indicate that the factor that most affected the response was the inoculum concentration (50.79% contribution), followed by the glucose concentration (25.22%), and shaking speed (14.79%). The contribution of temperature to the response variable was small (2.85%). This result was independent of the uncontrolled variation in the percentage of peptone in the sample…
With the strong growth in biologics, large molecules, and biopharmaceutical therapeutics in recent years, the pharmaceutical and biotech industries are increasingly turning toward peptides and proteins in the search for drug discovery targets. While both possess numerous properties that offer significant therapeutic potential, there are fundamental differences between the two compounds. This article examines some similarities and differences between proteins and peptides in light of potential market applications, manufacturing techniques, and the regulatory environment…
Back on March 13, 2009, this discussion question was posted to the Biotech and Pharma Professionals Network on LinkedIn: “What can industry do to encourage middle and high school students to pursue careers in biotech and pharmacology?” The response to this question was overwhelming. As of this writing, there are 1,467 comments posted. I have not read all of them yet. However, as a teacher of science at the high school level, I have been impressed by how involved network members have been by offering constructive suggestions to the industry intended to help encourage young students…