In today’s aggressive biopharmaceutical market, many drug discovery organizations, including both big pharmaceutical companies and small technology start-up companies, are outsourcing the development and manufacturing of their biopharmaceuticals to specialized contract manufacturing organizations (CMOs). Outsourced biopharmaceuticals range from those in early phase production to products that are well advanced down the development pipeline. As a result, there has been an expansion of CMOs that specialize in all aspects of biopharmaceutical manufacture, from process invention and development, through small-scale GMP production, to process validation and large-scale manufacture. The CMOs provide R&D services, quality function, and state-of-the-art good manufacturing practice (GMP) facilities needed for the production of biopharmaceutics. Using CMOs for biopharmaceutical process development and manufacture provides major cost savings by dispensing with the need to invest in experienced personnel and expensive manufacturing facilities…
BioProcessing Journal Posts
Baculovirus, particularly AcMNPV (Autographa californica multiple nucleocapsids polyhedrosis virus), is widely used for heterologous protein expression. There are several shortcomings in the current practice of preserving and scaling up baculovirus: 1) extracellular baculovirus stocks, routinely prepared in large volumes and stored at 4Âş C, are often unstable; 2) laborious and time-consuming steps to amplify and titer the baculovirus stocks are often necessary, and generally recommended, for achieving consistent viral infection and protein expression; 3) once prepared, the baculovirus is suspended and stored in conditioned medium. Given the complex, undefined, and unstable nature of the spent media components, including proteases and nucleases, protein expression tends to vary even when steps are taken to titer the virus stock and adjust the amount of stock used for infection. Here, we will report a new method for preserving and scaling up baculoviruses that: 1) provides a new form of viral stock more stable than the traditional, extracellular stock; 2) eliminates the need for virus amplification and retitering; 3) drastically reduces the turn-around time and resources required for scale-up; and 4) improves yield and consistency in protein expression.
Baculovirus infection of insect cells is an established method to obtain large quantities of biologically active recombinant proteins with properties similar to those of proteins expressed in mammalian cells. Insect cells are capable of most mammalian posttranslational modifications that control protein compartmentalization, secretion, targeting to nucleus or cell surface, N- and O-glycosylation, phosphorylation, proteolytic processing, and assembly of multi-protein complexes. The baculovirus transfer plasmids and accessory products utilized for protein expression in insect cells are currently available from several commercial sources. The plasmids often contain his-tags to facilitate Ni-NTA purification of recombinant proteins, and/or signal peptides to promote or enhance secretion of the proteins into the medium. Typically, in most baculovirus cloning vectors the coding region of the polyhedrin gene has been replaced by a polylinker with multiple cloning sites for the insertion of the cDNA of interest downstream of the strong polyhedrin promoter…
The Vmax™ technique has been used extensively to estimate filter area requirements for normal flow filtration (NFF) processes in biopharmaceutical applications. The benefits that this technique presents over conventional flow decay methods are the speed of testing, reduced volume requirements for evaluation, and competitive testing of varying filter types/sizes — all of which present an optimized filter screening strategy and preliminary estimate of optimized filter size requirements. Filter size or filtration area requirements derived using the Vmax technique consist of contributions from both capacity and flow-time aspects of the filtration process. This article examines the relative contributions of these terms to overall filter sizing vis-Ă -vis the ease of fluid filterability…
As pharmaceutical and biopharmaceutical companies become more global in nature with products that have the potential to reach into the worldwide marketplace, a special understanding is needed of the requirements that are specific to varying geographical areas. Specifically, the regions for worldwide pharmaceutical distribution can be broken into America, Europe, and Asia-Pacific, with each region presenting its own regulation and technical challenges. There are many issues that are common among these regions, but each region’s focus may be different. Typically, an issue arises in one region and then migrates to another as people become aware of the issues and concerns. For example, the use of prefilled syringe systems in Europe and Asia has migrated to the American marketplace, amounting to a more significant volume…
The diversity of the antibody-antigen interaction and our ability to manipulate this interaction has created an enormous potential for the discovery and development of IgG therapeutics and diagnostics. Along with the expanding clinical pipeline of antibody products, increasing efforts have been devoted to improving antibody production and purification procedures. In order to meet drug discovery needs with limited resources, the so called “flexible generic purification scheme” approach has been adopted to develop a robust manufacturing process that allows the application of similar operational conditions to different monoclonal antibody molecules…
Biopharmaceutical manufacturers are constantly seeking new ways to lower production costs, while simultaneously increasing cost effectiveness without sacrificing quality. The U.S. biotech industry has grown from $8 billion in 1992 to $30 billion in 2002. As productivity in biopharmaceutical manufacturing has increased, pressures to contain costs have mounted in the healthcare industry, coupled with increased demands by investors, which results in increased cost containment pressures on the industry as a whole. Some biotechnology products need to be produced in large quantities (hundreds of kilograms per year) to meet both current and expected demand. This requires significant manufacturing capacity, and makes the types of incremental process improvements commonly sought in chemical pharmaceutical processing an attractive proposition for biopharmaceutical manufacturing…
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…
Since the mid-1970’s, when Kohler and Milstein first discovered the process by which myeloma cells and splenocytes could be fused to produce monoclonal antibodies (MAbs), a whole new world of important therapeutic, prophylactic and diagnostic products has opened up, bringing in huge benefits for patients and manufacturers. The total sales of therapeutic MAbs reached more than $13 billion in 2005. Sixteen of the 18 FDA-approved MAbs came to the market after 1997, and over 150 are currently in clinical development, suggesting their increasing medical importance and the remarkable, recent advancements in development technology…