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…
Tag: <span>adenovirus</span>
Through the tremendous efforts of the Adenovirus Reference Material Working Group (ARMWG), an adenovirus reference material (ARM) is now available from the American Type Culture Collection (ATCC). The history and progress of the ARM production and characterization has been presented at many meetings and published in numerous journal articles. Although general statements have been made regarding how the ARM should be used, there is no formal directive or specific set of instructions detailing its application in the field. The goals of this paper are (1) to briefly review the objectives for development and implementation of the ARM, (2) to describe a critical assumption necessary to meet those objectives, (3) to outline specific approaches for using the ARM, and (4) to highlight the need for a working group to address the issues raised in the process…
Recombinant adenovirus are attractive as vectors for gene therapy because: they exhibit wide tissue tropism and high transduction efficiency; adenovirus cultures can reach high specific titers (10^10 VP/mL), and; their use in the treatment of cancer and other serious diseases is valuable. A primary mode of adenovirus purification continues to be CsCl density gradient centrifugation…
Production of non-enveloped viruses generally requires a cell lysis procedure to liberate mature particles trapped within their host cells. The standard bench-scale practice of using freeze/thaw cycles is simple and effective, but heat transfer limitations restrict the technique to relatively small applications. Here we show that a ten-minute treatment with a dilute mixture of polysorbate-80 and tri-butyl phosphate effectively liberates adenovirus from host cells…
As development proceeds for adenoviral vectors in gene transfer clinical trials, it becomes increasingly important that these products demonstrate a good safety profile, and thereby build confidence in those who must make decisions about risk/benefit ratios, dose escalation, and efficacy. Currently, safety and efficacy are based predominantly upon the analysis of data generated by non-standardized methods, resulting in inconsistent values being reported for virus titer and particle counts…
The Adenovirus Reference Material (ARM) was developed under the guidance of the Adenovirus Reference Material Working Group (ARMWG) and the U.S. Food and Drug Administration (FDA), and was made possible through the donation of services and supplies by a large number of laboratories and institutions from the United States, Canada, France, The Netherlands, Germany, and the United Kingdom. The purpose of the ARM is to provide a reference material for use in validating assays and internal standards for adenoviral particle concentration and infectious titer. The NIH Recombinant DNA Advisory Committee recommended the development of such a reference-testing agent in their report issued January 2002. The ARM consists of purified wild type 5 Adenovirus as described by ATCC’s catalog number VR-5…
The development of reference testing reagents has been used successfully in the past to standardize measurements among laboratories, particularly for biological products such as recombinant cytokines. This approach was recommended by many parties with a stake in adenovirus vector delivery in order to address the fact that particle units and infectious units are not standardized in the field. This has made interpretation of preclinical and clinical data, as it relates to the amount of adenovirus vector administered, difficult to compare across the field. An Adenovirus Reference Material is being developed to define the particle unit and infectious unit for adenovirus gene vectors, and create a commonality for comparisons, especially for data related to vector safety…
Introgen Therapeutics has been producing clinical-grade adenoviral vectors in scaled-up processes, in cGMP facilities, for over five years. Semi-automated hand filling, using a Watson-Marlow 505Di/L pump, has been used over this period to fill batch sizes of up to 2 liters of adenovirus. While this procedure has been robust and demonstrated a high level of sterility assurance through regularly scheduled media fill studies and product testing, the firm needed to move to the next level of fill sizes. Anticipating up to 10,000 fills in 3 mL vials, Introgen has worked in collaboration with M&O Perry Corp. to develop an automated fill capability that utilizes the same base procedure but in an automated fashion…
A formulation for purified adenoviral vectors was developed that provides stability through freeze-thaw stress and long-term storage at non-frozen temperatures. To evaluate the various test conditions, a panel of stability indicating methods was assembled, which included laser light scattering, HPLC, and transgene expression assays. Preformulation studies were conducted, and the effects of buffer species, pH, cryoprotectants, and salts upon adenoviral vector stability were determined…