recombinant proteins – Page 3 – BioProcessing Journal

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

A Flow Cytometric Assay for Rapid, Accurate Determination of Baculovirus Titers

Recombinant protein expression using the Baculovirus Expression Vector System (BEVS) is a powerful tool for the production of therapeutics, diagnostics, and reagents. To maximize efficiency of protein production, and thereby reduce costs, it is important to optimize the production parameters. A crucial step in optimization is determining the best multiplicity of infection (MOI) for the system in use. Factors that can affect the MOI include the recombinant baculovirus itself as well as cell line type and media composition. Typically the titer of a viral stock is determined in a standard manner, and then that titer is applied to each and every parameter tested; for instance, titering the virus on a Spodoptera cell line in a serum-containing media, and then using those data to determine the MOI used to infect Trichoplusia cells in a serum-free media formulation. The results may suggest that either the Trichoplusia cell line or the media formulation is inadequate for protein expression when, in fact, the MOI was incorrect for that particular combination…

Baculovirus Expression Technology Biologics Production

BEVS flow cytometry high-throughput optimization protein expression recombinant proteins viral titer

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

Encapsulating Cells for High-Productivity Recombinant Protein Expression

The first use of mammalian cell culture for the production of vaccines dates back to polio vaccine development in the 1950s. The development of hybridoma technology in the 1970s further stimulated the use of mammalian cells for the production of monoclonal antibodies. Together with developments in genetic engineering, it therefore became possible to produce a wide range of recombinant proteins as well as to improve cell metabolism. Animal cells are now widely used in industrial processes to obtain complex glycoproteins with correct posttranslational modifications and biological activity for diagnostic and therapeutic applications. Animal cells are the main source for commercially available recombinant proteins such as tissue plasminogen activator (tPa), erythropoietin (EPO), DNAse, factor VIII, interferon-ß, and monoclonal antibodies…

Biologics Production

continuous process encapsulation mammalian cell culture microcapsules non-anchorage-dependent cells protein expression recombinant proteins

Optimisation of Virus Stock Production and Cell Infection Conditions for Protein Expression

Variation of viral titre and recombinant product yields reported for the baculovirus expression vector system have been attributed to many specific infection variables. These include multiplicity of infection (MOI) and cell density at time of infection and time of harvest, as well as virus bank quality and efficiency. The MOI is defined as the number of plaque forming units (pfu) per cell that are added at the time of infection. Virus titre (pfu/ml) is determined by the plaque assay method. The MOI parameter is easily manipulated and may be important in optimising recombinant protein yields. Other sources of variation during both cell growth and viral infection phases may be responsible for the range of reported yields. Past studies in our laboratory compared the behaviour of cells infected with high and low MOI values, specifically regarding nutrient limitation and deprivation. In addition to these aspects, the quality of the virus bank may be an important factor which influences heterologous protein yields in the insect cell baculovirus system. Thus, production yields may be correlated to virus efficiency…

Baculovirus Expression Technology Biologics Production

BEVS protein expression recombinant baculovirus recombinant proteins viral titer virus stock

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

2003: The Year in Review

In general, the industry has gone through another of its realignment periods, where much was learned, but a lot of restructuring and refocusing took place. Driven by the need to keep the doors open, small to medium sized firms had to do some severe belt tightening, or completely redefine themselves as to technologies, products, and personnel. Many of the larger firms reevaluated their product pipelines, and then made the changes they felt were necessary to assure future revenues, or to make themselves attractive merger partners. Numerous large mergers took place with some that were the largest the biopharmaceutical industry has ever seen. In addition, several medium-sized companies merged, or otherwise found strategic alliances that energized their product pipelines, or simply provided the cash they needed to keep going. Antibody products did very well with a number of blockbusters receiving license approval in 2003…

Biologics Production

antibody therapeutics BEVS contract manufacturing facility design insect cell technology raw materials recombinant proteins transgenic production

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

Production of a Recombinant Influenza Vaccine Using the Baculovirus Expression Vector System

Influenza is a highly contagious, acute viral respiratory disease that occurs seasonally in most parts of the world. The infection resides primarily in the respiratory tract (nose, throat and bronchi), but causes both local and systemic symptoms including fever, chills, cough, headache, myalgia, sore throat, and malaise. Influenza-related pneumonia is the main complication of infection. Annual epidemics cause significant morbidity and mortality worldwide. Each year, influenza infections result in an average of 110,000 hospitalizations, approximately 20,000 of which result in death. These deaths are heavily concentrated (>90%) among persons who are at highest risk for influenza-related complications — elderly adults (over 65), children under age five, patients with pre-existing respiratory or cardiovascular disease, and women in the third trimester of pregnancy. Thus, the prevention of influenza virus infection is a major public health priority…

Baculovirus Expression Technology Viral Vectors

BEVS influenza purification recombinant proteins transfection trivalent vaccine vaccines

Advanced Methods of Adenovirus Vector Production for Human Gene Therapy: Roller Bottles, Microcarriers, and Hollow Fibers

Various types of viral vectors are being employed extensively as gene therapeutics to treat cancer and genetic diseases. Among the viruses that have been produced for human clinical trials (i.e. retrovirus, adenovirus, poxvirus, adeno-associated virus, and herpesvirus vectors) adenoviruses exhibit the lowest pathogenicity yet still infect an extensive range of cell types with high efficiency. These key characteristics make recombinant adenoviruses efficient gene-delivery vehicles and excellent research tools. However, the time-consuming and complex processes of generation, amplification, purification, and quality testing associated with production of recombinant adenoviruses make it difficult for many researchers to utilize these vectors. This is particularly true with respect to cell culture optimization and the virus propagation protocols employed in vector production. In this regard, the development of innovative cell culture techniques has become vital for optimizing vector production for gene therapy…

Biologics Production Cell & Gene Therapy Viral Vectors

adenoviral vectors analysis cell culture enumeration propagation purification recombinant proteins

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