scholarly journals Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer

2021 ◽  
Vol 9 ◽  
Author(s):  
Elise K. Mullins ◽  
Thomas W. Powers ◽  
Jim Zobel ◽  
Kory M. Clawson ◽  
Lauren F. Barnes ◽  
...  
Keyword(s):  
Viral Particle ◽  
Analytical Ultracentrifugation ◽  
Product Yield ◽  
Initial Assessment ◽  
Genome Packaging ◽  
Charge Detection Mass Spectrometry ◽  
Charge Detection ◽  
Differential Infectivity ◽  
Multiple Species

We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. To determine a possible reason for this outcome, we characterized the proportion and composition of the empty and packaged capsids. Both analytical ultracentrifugation (AUC) and differential centrifugation sedimentation (DCS, a rapid and quantitative method for measuring adenoviral packaging variants) were employed for an initial assessment of genome packaging and showed multiple species whose abundance deviated between the virus builds but not manufacturing campaigns. Identity of the packaging variants was confirmed by charge detection mass spectrometry (CDMS), the first known application of this technique to analyze adenovirus. The empty and packaged capsid populations were separated via preparative ultracentrifugation and then combined into a series of mixtures. These mixtures showed the oft-utilized denaturing A260 adenoviral particle titer method will underestimate the actual particle titer by as much as three-fold depending on the empty/full ratio. In contrast, liquid chromatography with fluorescence detection proves to be a superior viral particle titer methodology.

scholarly journals Assessment of genome packaging in AAVs using Orbitrap-based charge detection mass spectrometry

2021 ◽  
Author(s):  
Tobias P Woerner ◽  
Joost Snijder ◽  
Olga Friese ◽  
Thomas W Powers ◽  
Albert J.R. Heck
Keyword(s):  
Mass Spectrometry ◽  
High Demand ◽  
Genome Packaging ◽  
Charge Detection Mass Spectrometry ◽  
Charge Detection ◽  
Essential Step ◽  
Gene Therapy Vectors ◽  
Effective Drugs ◽  
Limited Sensitivity

Adeno-associated viruses (AAV) represent important gene therapy vectors with several approved clinical applications and numerous more in clinical trials. Genome packaging is an essential step in the bioprocessing of AAVs and needs to be tightly monitored to ensure the proper delivery of transgenes and the production of effective drugs. Current methods to monitor genome packaging have limited sensitivity, a high demand on labour, and struggle to distinguish between packaging of the intended genome or unwanted side-products. Here we show that Orbitrap based charge detection mass spectrometry allows the ultra-sensitive quantification of all these different AAV bioprocessing products. A protocol is presented that allows the quantification of genome packed AAV preparations in under half an hour, requiring only micro-liter quantities of typical AAV preparations with ~1013 viral genome copies per millilitre. The method quickly assesses the integrity and amount of genome packed AAV particles to support AAV bioprocessing and characterization of this rapidly emerging class of advanced drug therapies.

scholarly journals Resolving Adeno-Associated Viral Particle Diversity With Charge Detection Mass Spectrometry

2016 ◽  
Vol 88 (13) ◽  
pp. 6718-6725 ◽  
Author(s):  
Elizabeth E. Pierson ◽  
David. Z. Keifer ◽  
Aravind Asokan ◽  
Martin F. Jarrold
Keyword(s):  
Mass Spectrometry ◽  
Viral Particle ◽  
Charge Detection Mass Spectrometry ◽  
Charge Detection

Acute Traumatic Cervical Facet Fractures

2018 ◽  
Vol 1 (2) ◽  
pp. 5
Author(s):  
Shankar Gopinat
Keyword(s):  
Spinal Cord ◽  
Cervical Spine ◽  
Early Surgery ◽  
Initial Assessment ◽  
Trauma Patients ◽  
Spinal Stability ◽  
Fracture Patterns ◽  
Spine Ct ◽  
Cervical Facet

Acute cervical facet fractures are increasingly being detected due to the use of cervical spine CT imaging in the initial assessment of trauma patients. For displaced cervical facet fractures with dislocations and subluxations, early surgery can decompress the spinal cord and stabilize the spine. For patients with non-displaced cervical facet fractures, the challenge in managing these patients is the determination of spinal stability. Although many of the patients with non-displaced cervical facet fractures can be managed with a cervical collar, the imaging needs to be analyzed carefully since certain fracture patterns may be better managed with early surgical stabilization.

scholarly journals Advances in LC–MS/MS Methods for Allergen Testing, Meat Speciation, and Gelatin Speciation

2019 ◽  
Vol 102 (5) ◽  
pp. 1309-1315
Author(s):  
Jianru Stahl-Zeng ◽  
Ashley Sage ◽  
Philip Taylor ◽  
Jeremy Dietrich Netto ◽  
Tuo Zhang
Keyword(s):  
Mass Spectrometer ◽  
Multiple Reaction Monitoring ◽  
Proteome Analysis ◽  
Food Product ◽  
Transition Analysis ◽  
Tandem Quadrupole Mass Spectrometer ◽  
Signature Peptides ◽  
Unique Markers ◽  
Multiple Species

Abstract Background: Food authenticity is demanded by the consumer at all times. The consumer places trust in the manufacturer that the food product is genuine in terms of what is recorded on the packaging label. Objective: Recent advancements in LC–tandem MS methodology in the detection of allergens, meat, and gelatin speciation in raw food products and processed foods are detailed in this paper. Method: For each of the three methods, initial proteome analysis and the screening leading to the determination of unique tryptic peptides were conducted using a high-resolution, accurate tandem mass spectrometer. Having identified the unique markers, the method was transferred to a tandem quadrupole mass spectrometer for a higher-sensitivity quantitative study, multiple reaction monitoring transition analysis. Results: For the allergens method a detection limit of at least 10 ppm was attained across the 12 allergen peptides in this workflow. In the gluten workflow the resulting chromatograms show good detection down to 5 ppm, with no interference from the food matrices. The meat speciation method details that signature peptides could be readily identified at 1% w/w with no matrix interference. Conclusions: These single-injection workflows with cycle-time optimization enable wide coverage of analytes to identify multiple species within challenging matrix samples.

Sign in / Sign up

Export Citation Format

Share Document