Mammalian cell culture processes used in the production of monoclonal and recombinant proteins require orthogonal steps in the downstream process to clear virus. Virus filtration is a common, size exclusion-based method in these processes. Initially, tangential flow products were used, but as a result of new-generation virus filtration products, most operations are now constant-pressure, normal-flow operations that can be executed in a single shift operation. The virus filtration step toward the end of the downstream process is now yielding increased protein concentrations…
Tag: <span>recombinant proteins</span>
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…
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…
In the last few decades, laboratory and therapeutic applications of cell culture-derived biologicals have expanded from their use in diagnostic and research fields to the prevention and treatment of infectious diseases, certain forms of cancer, immunological and congenital conditions, and cell and gene therapy. While significant therapeutic benefits obtained from the use of cell culture-derived biologics (e.g., recombinant proteins, monoclonal antibodies [mAb], and vaccines) are unequivocal, the complexities associated with the manufacture of such products is acknowledged. Primary and continuous cell lines used in the manufacture can be associated with risk of contamination with endogenous retroviruses, latent viruses, or new and emerging agents. Some cell lines, such as Chinese hamster ovary (CHO) cells, have an excellent safety record with no documented safety risks…
Human granulocyte colony-stimulating factor (GCSF) is produced by biotech laboratories and production facilities for reducing neutropenia duration and sequels in patients with myelosuppressor treatments, among other applications. However, real challenges for these laboratories started in 2015 when the PEGylated-GCSF patent expired, opening alternatives for developing biomanufacturing processes and new applications. Thus, the purpose of this study was to analyze downstream process controls designed to ensure recombinant human GCSF (rh-GCSF) quality and to provide some evidence of the downstream process validation status. Study outcomes proved that the rh-GCSF expression system was stable and chromatographic profiles were reproducible among samples.
Nowadays, therapeutic monoclonal antibodies (mAbs) are predominantly produced with mammalian cell culture systems such as those using Chinese hamster ovary (CHO) cells. Efforts are underway to reduce the costs of this process to meet the increasing global demand in biopharmaceuticals; meanwhile, cheaper and faster expression systems are being investigated as alternatives. The yeast, Pichia pastoris, has become a substantial workhorse for recombinant protein production. However, the N-linked glycosylation in P. pastoris, namely high mannose glycosylation, is significantly different from that in CHO or other mammalian cells, including human cells. In this study, a SuperMan5 strain of P. pastoris was constructed using Pichia GlycoSwitch® technology to successfully produce a more mammalian-like immunoglobulin G (IgG) fragment crystallizable (Fc), which showcases the potential of P. pastoris as a next-generation mAb production platform. Importantly, in this study, a strong methanol-independent promoter, PUPP, was applied, which only requires glycerol feeding for protein production. Most P. pastoris promoters used for protein expression are derived from genes in the methanol metabolism pathway, creating safety concerns due to the flammable nature of methanol, especially at large scale. Here, a fed-batch SuperMan5 P. pastoris fermentation was carried out in which methanol induction, as well as its affiliated safety risks, were eliminated. Overall, this study provides insights into the development of safe and cost-effective industrial mAb production approaches independent of mammalian cell culture.
Stirred tank single-use bioreactors (SUBs) have been widely adopted for production of biopharmaceuticals such as monoclonal antibodies in mammalian cell culture. However, they are seldom used for commercial production of biologics with microbial fermentation. SUBs offer time-saving advantages because they do not require significant downtime for cleaning and sterilization, so finding a SUB that can perform well with high cell density microbial fermentation processes has the potential to increase the number of production runs. Therefore, for this study, a His-tagged protease inhibitor was chosen as a model protein to demonstrate that the Sartorius Biostat STR® MO, a SUB recently developed for microbial fermentation, is suited for recombinant protein production by high cell density Escherichia coli fermentation processes.
At 50 L scale, the SUB achieved good process control and allowed an oxygen uptake rate (OUR) of up to 240 mmoles/L/h. The fermentation runs produced up to 5.8 g/L of the soluble recombinant protein and a dry cell weight of >60 g/L at the end of fermentation. Additionally, the SUB showed a similar fermentation profile when compared with data from parallel runs in 15 L sterilise-in-place (SIP) vessels using identical media and process parameters. This study indicates that with a minimum investment of capital resources, stirred tank SUBs could be used in pilot-scale manufacturing with high cell density microbial fermentations to potentially shorten the timelines and costs of advancing therapeutic proteins to clinic.
In general, yeasts offer advantages for recombinant protein expression because their intracellular environment is suitable for the correct folding of recombinant proteins and grow very high cell densities in defined fermentation media. Within the yeast kingdom, Pichia pastoris has been successfully used for expressing several recombinant proteins. The genome of this yeast contains two copies of the alcohol oxidase (AOX) gene, where the AOX1 promoter regulates 85% of the alcohol oxidase activity and drives the recombinant protein expression into the cell. One of the most successfully recombinant proteins expressed in Pichia pastoris is the hepatitis B surface antigen (HBsAg). The current manufacturing process of the active pharmaceutical ingredient (HBsAg) of the Cuban hepatitis B vaccine (HeberBiovacâ„¢ HB) starts with the expression of the HBsAg in Pichia pastoris….
Heterologous expression of membrane proteins remains a bottleneck for structural characterization by x-ray crystallography. Such proteins represent approximately 30% of the proteome and are not sufficiently represented in the Protein Data Bank (PDB). G-protein-coupled receptors (GPCRs) are an area of particular interest as it is estimated that one third of current FDA approved drugs act through this class of receptors. We have been studying rhodopsin with an interest in determining the conformational change that leads to signal transduction in this class of receptors. Although there has been some success in expressing select members of the large GPCR family in bacterial systems, the best characterized expression systems have generally been in mammalian tissue culture…
The recombinant protein alefacept (Amevive®) is an immunomodulator approved for the Âtreatment of moderate to severe chronic plaque psoriasis. In phase 3 clinical studies, treatment of psoriatic patients with alefacept at a safe and well-tolerated dose caused Âmeaningful reductions in psoriasis area and severity index and in physician global Âassessment scores as well…