Adenoviral vectors for gene delivery are being tested in the clinic for a number of indications and therapeutic uses. In order to facilitate the comparison of studies from different laboratories, the Adenovirus Reference Material Working Group (ARMWG) has developed a reference testing reagent, which will be referred to as the Wild Type Ad5 Adenoviral Reference Material (ARM). This ARM will allow laboratories to standardize in-house controls employed in assays for the determination of particle concentration and infectious titer of their own adenoviral preparations. As part of this project, short-term field use and shipping studies were performed on the ARM. The virus was found to be stable under simulated shipping conditions, for one thaw after shipping, and at 4 °C for up to four hours after thawing. However, there was evidence of aggregation in some vials with repeated freeze-thaw cycles. Therefore, we recommend that each vial be treated as a single-use aliquot, and that it be used within four hours of thawing…
BioProcessing Journal Posts
The insect cell/baculovirus expression system typically results in more rapid expression and higher concentrations of recombinant proteins than what can be achieved with other animal cell culture systems. The lack of complex glycosylation in the proteins produced by this system, however, limits its use in the commercial-scale production of therapeutics. Complex glycosylation is required in many cases for adequate protein activity and pharmokinetic characteristics. In contrast to the protein’s primary structure, which is encoded by the genetic material and is constant regardless of the host utilized, the extent of glycosylation is determined by the host, and by the protein itself. Even cells from different tissues of the same organism provide different glycosylation profiles. In addition, culture conditions and the cellular metabolic state can also influence protein glycosylation…
Based on feedback received from a number of our recent conferences, cell culture media development remains one of the biggest challenges in the development of biological products. With more products reaching larger production scale and licensed production, it is becoming ever more important that we gain a better understanding of the media supply industry, and that we find ways to make media development more economical, reliable, and reproducible…
By virtually any measure, constraints in current manufacturing capacity are hindering the development of new biologic drugs, as well as the greater market penetration of several licensed biologics. This capacity demand is being driven not only by the increasing number of new biologics being approved, but by the number of biologics that are in the product development pipeline. Figure 1 shows United States FDA biologics approvals for the 20-year period from 1981-2000. While there is year-to-year variability in approvals, especially in later years, the five-year averages show a doubling in the annual rate of product approval for each successive five-year period. Clearly, these averages cannot continue to increase at the same rate. In fact, only six biologics were approved by the FDA in 2001…
The baculovirus expression vector system (BEVS) is one method utilized for the production of recombinant proteins, and typically works without significant difficulties. However, some proteins are produced in insoluble forms, and degradation can occur. This article will focus on this degradation issue, and present a method to stabilize a protease-sensitive protein that has been produced at the 40-liter scale…
Growth media for mammalian cell culture are complex mixtures of raw materials that include amino acids, vitamins, inorganic salts and a wide variety of other components. The risk of infectious agent transmission, when some of these components are derived directly from animals, is a major concern in the biopharmaceutical industry, and is being actively addressed. However, the risk associated with the use of indirectly, or secondarily, derived animal components is less recognized and addressed. We have developed a classification system to define the contact level that a cell culture medium component has had with animal-derived materials. This classification system has increased the accuracy and reliability of the information we are able to obtain from raw material manufacturers, and is being used as part of a risk assessment analysis for a serum/protein-free media we are moving from development into manufacturing…
Serine-threonine kinases of the Mitogen Activated Protein Kinase (MAPK) pathway represent potential drug targets for a wide range of diseases. As part of an effort to understand the biology of the pathways, several human serine-threonine MAPKs were produced. Optimization and modification of current methodologies used in the baculovirus expression system resulted in the generation of large amounts of active MAPKs. Compounds found to inhibit the MAPKs in vitro, subsequently showed activity in cell-based assays and animal models. The processes to be discussed were developed to yield large quantities of three active human serine-threonine MAPKs by the baculovirus expression system…
The baculovirus expression system promises to revolutionize the production of recombinant proteins for use as clinical products. The technology is robust, efficient, and low-cost when compared to other cell based systems. The technique may also present an advantage in producing safer products versus the equivalent materials made with mammalian cells. Proteins can be produced in insect cells without animal supplements such as fetal calf serum. In the current climate of concerns over Bovine Spongiform Encephalopathies, and bovine viral risks, this method offers a significant safety, as well as cost advantage, over other production methods…
Globally, an estimated 36 million people are living with HIV, and some 20 million people have already died of AIDS. Today, there is still no HIV vaccine available. HIV virus-like particles are an attractive vaccine candidate due to their ability to induce both antibody and cytotoxic T-lymphocyte responses. In this article, we describe the development of a production process for an HIV particle vaccine, HIV-1 p55 (gag). The gag precursor protein (p55) is sufficient for assembly and cellular release of retrovirus-like particles. We expressed the p55 gag protein using the BEVS technology in Spodoptera frugiperda expresSF+ cells…