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Purification of Recombinant Baculovirus by End-Point Dilution and gp64 Screening

by Brooks Hayes and Thera Mulvania, PhD
Volume 12, Issue 3 (Fall 2013)

The baculovirus expression vector system (BEVS) has emerged as a powerful tool for the production of recombinant proteins used as therapeutics, reagents, and diagnostics. In order to maximize the system’s efficiency and thereby reduce costs, optimizing production parameters is imperative. A critical factor in optimization is the production of a high-quality baculovirus stock with a high-titer, pure clonal population of recombinant virus that is stable over time. Baculovirus stocks may contain alternate varieties of infectious virus due to cross-contamination, outgrowth of non-recombinant virus, and excision of inserts attributable to some recombinant virus production technologies. Since the advent of the BEVS, the “gold standard” for production of pure baculovirus stocks has been plaque purification. Briefly, plaque purification involves infecting a monolayer of cells with dilutions of virus before applying an agarose overlay to the monolayer. After a 5–7 day incubation, isolated plaques can be picked, virus eluted from the agarose plug, and amplified. The drawbacks of plaque purification are: (1) it is time and labor-intensive; (2) the results hinge greatly on the health of the cells and the cell density at infection; (3) identification and picking of isolated plaques is challenging; and (4) the integrity of the procedure is easily compromised by virus diffusion and mass flow of virus-containing liquid beneath the agarose overlay...

Citation:
Hayes B, Mulvania T. Purification of Recombinant Baculovirus by End-Point Dilution and gp64 Screening. BioProcess J, 2013; 12(3): 20-27.
http://dx.doi.org/10.12665/J123.Hayes.

Posted online September 30, 2013.

 
Model and Method for Optimal Sizing of Serial Microfiltration Systems

by Sal Giglia and Greg Straeffer
Volume 12, Issue 3 (Fall 2013)

Classical filtration fouling models do not accurately predict fouling behaviors for sterilizing filtration applied to some bioprocess fluids, particularly when filters are operated in series. To overcome the limitations of these models, we extended a previously developed single-stage fouling model that combined blocking and adsorption mechanisms to dual filters operated in series. Our model demonstrates improvements in predicting bioprocess fluid filtration performance accuracy when applied to microfiltration membranes operated in series, from measured performance of each membrane filter operated individually. In addition, the model and developed method allows for rapid and efficient optimization of prefiltration to final filter area ratios. In redundant sterile filtration, two filters with the same pore size rating are operated in series to protect against an integrity failure of the primary sterilizing filter. Prefilters can also be used to protect final sterilizing-grade membrane filters from excessive fouling which in turn offer the benefits of reduced costs and filtration time...

Citation:
Giglia S, Straeffer G. Model and Method for Optimal Sizing of Serial Microfiltration Systems. BioProcess J, 2013; 12(3): 31-39.
http://dx.doi.org/10.12665/J123.Giglia.

Posted online September 30, 2013.

 
Investigation of Light Isomerization of Tropolone Using Liquid Chromatography and Orbitrap Mass Spectrometry

by Robert J. Duff, PhD and John L. Snyder, PhD
Volume 12, Issue 3 (Fall 2013)

This investigation into the light isomerization of tropolone was initiated after a method for determining residual tropolone in the bulk drug substance (BDS) of a therapeutic monoclonal antibody was developed and validated at our laboratory. This method, developed at a client’s request, required a detection limit of 50 parts per billion (ppb) in the BDS to meet their regulatory requirements. The method was developed using liquid chromatography and tandem mass spectrometry (LC/MS/MS) because of the selectivity and sensitivity of the technique when using selective reaction monitoring (SRM). During development, however, it was discovered that only after creating a photoisomer of tropolone could successful separation, detection, and quantitation of tropolone be achieved. During validation, the method showed good performance...

Citation:
Duff RJ, Snyder JL. Investigation of Light Isomerization of Tropolone Using Liquid Chromatography and Orbitrap Mass Spectrometry. BioProcess J, 2013; 12(3): 10-19.
http://dx.doi.org/10.12665/J123.DuffSnyder.

Posted online September 30, 2013.

 
Myriad Further Limits Patent Eligibility

by William K. Merkel, PhD, JD
Volume 12, Issue 2 (Summer 2013)

The recent decision of the US Supreme Court in Assoc. Mol. Pathol. v. Myriad Genetics, Inc., No. 12-398 (June 13, 2013) continued the Court’s efforts to clarify those innovations eligible for patent protection. Myriad unanimously held that “isolated” BRCA1/2 DNAs were patent ineligible under 35 USC § 1 01 while BRCA1/2 complementary DNAs (cDNAs) were patent eligible under that statutory provision. In considering patent eligibility, the Court proposed a test for isolated DNAs that balances incentives and impediments to innovation arising from patent protection, without placing any weight on whether the innovation relates to nature or to an abstract idea. The Court concluded that the mere act of isolating DNA segments claimed in terms of their genetic information was insufficient to render the claims patent eligible. Although the full effect of Myriad won’t be fully known for some time, it is already apparent that reliance expectations developed over 30 years have been upset. Taking a closer look, Myriad discovered the BRCA1 and BRCA2 genes and their influence on the risks of breast and ovarian cancers. Myriad also located these genes in the human genome, isolated the genes, and determined the sequences of several alleles, or versions, of these genes, with some alleles associated with higher cancer risks than others. This led Myriad to develop and market a diagnostic test for assessing breast and ovarian cancer risk, and to seek patent protection for the technology. Myriad refused to license the technology to competitors, which led to a declaratory judgment suit against Myriad. The district court granted summary judgment to the challengers, holding that Myriad’s isolated BRCA1/2 DNA claims and BRCA1/2 cDNA claims were invalid for patent ineligibility under 35 USC § 101. For reasons not relevant to the patent eligibility of DNA composition claims, the Court of Appeals for the Federal Circuit (CAFC) had two cracks at this case on appeal, and both times it held that isolated BRCA1/2 DNAs and BRCA1/2 cDNAs were patent eligible. On its own second review of the case, the US Supreme Court unanimously and finally decided the appeal of summary judgment, holding isolated BRCA1/2 DNAs patent ineligible, but BRCA1/2 cDNAs patent eligible...

Citation:
Merkel WK. Myriad Further Limits Patent Eligibility. BioProcess J, 2013; 12(2): 37-41.
http://dx.doi.org/10.12665/J122.Merkel.

Posted online August 1, 2013.

 
Adventitious Virus Contamination Testing: Massively Parallel Sequencing in a Multimodal Solution for Biopharmaceutical Safety Testing

by Jack X. Yu, Stephen Karakasidis, Ginger Zhou, Wenying Huang, Yankai Jia, Michael J. Hantman, PhD, and Jeffrey A. Shaman, PhD
Volume 12, Issue 2 (Summer 2013)

Virus contamination of commercially valued cell cultures can be a health risk to the general population and imposes financial burdens on manufacturing and biopharmaceutical companies. We investigated the use of massively parallel sequencing/next-generation sequencing (MPS/NGS) and bioinformatics for the detection and subsequent identification of adventitious contamination. Specifically, the Illumina® HiSeq 2000 instrument, in the 2 × 100 base pair (bp) paired end (PE) run configuration, was used to determine the identity and limit of detection of a DNA virus spiked into a virus vaccine, and an RNA virus spiked into a mammalian master cell bank (MCB). This configuration provided sufficient sequence read lengths and depth of coverage to detect and identify spiked-in SV40 and measles viruses in a background of vaccine, MCB, and host nucleic acid that make up the bulk of these samples. Furthermore, the detection of 30 SV40 reads within one tested sample suggested a < 1 plaque forming unit (pfu) sensitivity in a background of 2.8 × 107 infectious adenovirus serotype 5 (Ad5) particles. Results from subsequent virus vaccine testing suggested the presence of non-viable virus DNA contamination in the sample. Therefore, we propose that a multimodal approach, in which broad-range screening for known or unknown adventitious agents by MPS/NGS is complemented by targeted virus detection assays (e.g., PCR-based and infectivity assays), should provide the most useful safety monitoring information. We suggest that MPS/NGS and the accompanying bioinformatics is a sensitive, broad-range, and long-lasting tool with the ability to improve upon existing biosafety testing within a larger testing program...

Citation:
Yu JX, Karakasidis S, Zhou G, Huang W, Jia Y, Hantman MJ, Shaman JA. Adventitious Virus Contamination Testing: Massively Parallel Sequencing in a Multimodal Solution for Biopharmaceutical Safety Testing. BioProcess J, 2013; 12(2): 18-24.
http://dx.doi.org/10.12665/J122.ShamanHantman.

Posted online August 1, 2013.

 
A Proposed Modeling Approach for Comparing the Heat Inactivation Susceptibility of Viruses

by Raymond Nims, PhD and Mark Plavsic, PhD, DVM
Volume 12, Issue 2 (Summer 2013)

Heat inactivation is dependent both on temperature and time at temperature, making inter-assay and intervirus comparisons of heat sensitivity of viruses problematic. Historically, heat inactivation data for pathogens, including viruses, have been evaluated by determining decimal reduction value ([D] the time required to inactivate 1 log10 of the organism at a given temperature) and the incremental temperature required to decrease the D by 1 log10 (z). We recommend the use of a straightforward approach for extrapolating heat inactivation (i.e., inactivation vs. time at fixed temperature) data from measured to non-measured temperatures that is based not on the z value, but on a power function fit of the D vs. temperature plots. There needs to have been at least three temperatures evaluated in the inactivation vs. time kinetics studies in order to conduct these modeling analyses. For inter-assay and inter-virus comparisons of heat inactivation sensitivity, we propose the use of two modeled parameters: (1) temperature required to inactivate 1 log10 of virus in 0.5 minutes; and (2) time required for 1 log10 reduction in infectivity at 80 °C. By using both modeled parameters, we have calculated consensus heat inactivation values for two caliciviruses (feline calicivirus and murine norovirus)...

Citation:
Nims R, Plavsic M. A Proposed Modeling Approach for Comparing the Heat Inactivation Susceptibility of Viruses. BioProcess J, 2013; 12(2): 25-35.
http://dx.doi.org/10.12665/J122.Nims.

Posted online August 1, 2013.

 
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