therapeutic proteins – BioProcessing Journal

Study of a Single-Use Stirred Tank Bioreactor for Manufacturing a Therapeutic Protein in a High Cell Density Microbial Fermentation

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.

Manufacturing

fermentation microbial fermentation recombinant proteins single-use bioreactor stirred-tank bioreactor therapeutic proteins

Rapid Development of High-Performance Stable Mammalian Cell Lines for Improved Clinical Development

Since the first approval for human use of a recombinant protein therapeutic, this sector of the pharmaceutical market has grown rapidly. The first approved protein therapeutics were small, non-glycosylated proteins such as insulin and human growth hormone; they were produced in bacterial systems. With the advent of mammalian cell-based production systems, it became possible to produce more complex, glycosylated proteins for use as recombinant therapeutics…

Biologics Production

cell line selection mammalian cell line development recombinant proteins stability therapeutic proteins vector construction

Product Characterization of Recombinant Proteins and Monoclonal Antibodies

The analytical characterization of recombinant protein therapeutic drug products has broadened to include the use of more sophisticated technologies. The expansion of technical abilities has translated into increasing the depth and breadth of our knowledge and understanding of the drug product intended for commercialization. With the availability of more precise methods, the regulatory expectations for understanding the characteristics of a protein therapeutic drug product are increasing. A thorough understanding of a therapeutic protein’s biochemical and biophysical characteristics is necessary to support investigational new drug (IND) applications and other drug regulatory filings…

Regulatory

analysis characterization monoclonal antibody recombinant proteins standards therapeutic proteins

A Universal Platform for the Purification of Therapeutic Proteins Using Affinity Tags: The Use of Engineered Aminopeptidases for His-Tag Removal

Current expression technologies have enabled the production of thousands of recombinant proteins in diverse production hosts. Therapeutic recombinant proteins have been engineered for a variety of purposes including reduced antigenicity, longer half-life, simplified process development, and increased affinity. Protein engineering has relied on various high throughput methods (e.g., directed evolution, phage display) to identify candidate proteins with the desired therapeutic properties. The physiological and biochemical diversity of native and engineered proteins reflects on the abundance of production hosts, expression tools, and different approaches for protein purification. Notably, a key step in high-throughput protein production is purification, which is a bottleneck where large numbers of samples are involved. Universal purification methods that can be applied to virtually any protein, and that are amenable to automation, can be used to address this problem…

Biologics Production

affinity chromatography affinity tag aminopeptidase chromatography his-tag removal platform purification therapeutic proteins

A Novel Human Expression System for Generating Glycoprotein with Higher Activity, Fully Human Glycosylation, and Optimised Sialylation

Today concentrated efforts are underway to improve the bioactivity of therapeutic proteins with the aim of reducing: (i) the number and concentration of the applied doses of the therapeutic protein, (ii) undesired side effects, and (iii) the cost of a therapy. A very promising strategy is to optimise the glycosylation of these biotherapeutics. A novel expression platform, GlycoExpress™, has been developed to produce proteins with fully human glycosylation, optimised sialylation, and improved bioactivity…

Biologics Production

bioactivity expression glycosylation optimization scale-up sialylation therapeutic proteins

The Intraocular Delivery of Neuroprotective Factors to the Retina Using Encapsulated Cell Technology

Ophthalmic disorders are a group of diseases with a rapidly increasing frequency associated with an increase in the aged population. Patients with potentially blinding diseases have become one of the largest segments of the healthcare field, with more than 50 million patients in the United States alone. Their sight is threatened by diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma, or retinitis pigmentosa (RP). Until recently, there were essentially no effective treatment options to halt the progression of chronic, potentially blinding diseases. Biotechnological advances have resulted in the development of a variety of promising new protein factors that, if delivered to diseased cells of the retina, hold promise for treatment by interrupting or reversing the disease process…

Cell & Gene Therapy

cellular engineering encapsulated cell technology neurotrophic cytokines neurotrophic factor ophthalmic disorder therapeutic proteins

Optimizing Protein Expression: Producing Pure Protein for Structural Biology

Proteins and their promise for revolutionizing drug discovery have come virtually full circle in just a few decades. The advent of genetic engineering and the emergence of early recombinant proteins such as insulin and interferon dramatically boosted the perceived value of proteins in pharmaceutical research and of protein drugs in particular. Although the lights dimmed somewhat on the promise of therapeutic proteins in subsequent years, more recent times have seen a resurgence of interest in proteins, particularly monoclonal antibodies. Perhaps most telling has been the dawn of the post-genomic era, which has cast a bright spotlight on proteins, long respected as the work-horses of the cell, for their usefulness in exploring cell function, unraveling biochemical pathways, understanding disease, and for their massive value as novel drug targets…

Biologics Production

automation optimization protein expression purification recombinant proteins scale-up therapeutic proteins

Initial Clarification by Aqueous Two-Phase Partitioning of Leaf Extracts from Solanum tuberosum Plants Expressing Recombinant Therapeutic Proteins

The use of plants as protein expression hosts for human therapeutic proteins is emerging as a safe and cost-effective alternative to microbial and mammalian cell culture. Pharmaceutical protein production is typically carried out in microbes and mammalian cell culture because of their high production potential and/or ability to produce complex eukaryotic proteins. However, immense costs are typically required for production facilities to support their growth. To offset these costs, companies usually build and expand a production facility over several years. In fact, it has been predicted that the demand for high-value pharmaceuticals produced by cell culture will quickly surpass the ability of pharmaceutical companies to produce them…

Biologics Production

aqueous two-phase partitioning clarification plant-based protein production protein expression protein extraction recombinant proteins therapeutic proteins

Development of a High-Throughput Protein Expression Strategy

At the onset of modern-day biotechnology, products typically fell into two distinct categories, the traditional high volume, low value products (e.g. beer and industrial enzymes) that had come to characterize the biotechnology industry, and low volume, high cost products. Recombinant proteins, the result of technological advances in molecular biology, have come to typify these latter products. Recombinant protein therapeutics have been hugely successful, potentially outstripping production capacity and continue to drive much of the biotechnology. Meanwhile, many recombinant proteins, those characterized as research tools and reagents, are governed by a price-volume relationship typical of industrial enzymes. In a competitive environment, they are fast becoming commodities — price sensitive, packaged as kits, coupled to instrumentation, and relying on heavy marketing and brand recognition. Ominously, the advantage protein therapeutics have enjoyed with patent protection and regulatory constraints on production is being threatened as patents expire and competition from generics increases…

Biologics Production

automation characterization high-throughput protein expression purification recombinant proteins scale-up therapeutic proteins

Role of the Container/Closure System and Formulation on Agitation-Induced Aggregation Phenomena in Recombinant Adenoviral Products

The biologic activity of protein therapeutics is often compromised by the formation of soluble aggregates and protein precipitates. While the detailed molecular mechanism of protein aggregation remains unclear, substantial evidence suggests that factors that promote protein unfolding and exposure of hydrophobic residues play a significant role in aggregate formation. These can vary according to the particular protein of interest, the solvent system, and storage conditions. Slight changes in temperature, pH, and ionic strength have been documented to have a significant effect on the aggregation phenomena of numerous proteins. Freezing and subsequent thawing of a preparation (whether by design or accident) during processing, shipping, and storage can induce subtle changes in the ionic strength, solute concentration, and pH of a given preparation and induce aggregate formation…

Biologics Production Viral Vectors

adenovirus aggregation protein aggregation recombinant proteins therapeutic proteins

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