Preparation of pure, high titer, pseudoinfectious Flavivirus particles by hollow fiber tangential flow filtration and anion exchange chromatography

Vaccine ◽  
2015 ◽  
Vol 33 (35) ◽  
pp. 4255-4260 ◽  
Author(s):  
Sophia T. Mundle ◽  
Maryann Giel-Moloney ◽  
Harry Kleanthous ◽  
Konstantin V. Pugachev ◽  
Stephen F. Anderson
Keyword(s):  
Anion Exchange ◽  
Hollow Fiber ◽  
High Titer ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Tangential Flow Filtration ◽  
Tangential Flow ◽  
Flow Filtration

Purification of plasmid DNA using tangential flow filtration and tandem anion-exchange membrane chromatography

2008 ◽  
Vol 32 (5) ◽  
pp. 615-623 ◽  
Author(s):  
Patricia Guerrero-Germán ◽  
Duarte M. F. Prazeres ◽  
Roberto Guzmán ◽  
Rosa Ma. Montesinos-Cisneros ◽  
Armando Tejeda-Mansir
Keyword(s):  
Anion Exchange ◽  
Plasmid Dna ◽  
Anion Exchange Membrane ◽  
Tangential Flow Filtration ◽  
Tangential Flow ◽  
Membrane Chromatography ◽  
Flow Filtration ◽  
Exchange Membrane

scholarly journals Cytoplasmic tail truncation of SARS-CoV-2 Spike protein enhances titer of pseudotyped vectors but masks the effect of the D614G mutation

2021 ◽  
Author(s):  
Hsu-Yu Chen ◽  
Chun Huang ◽  
Lu Tian ◽  
Xiaoli Huang ◽  
Chennan Zhang ◽  
...  
Keyword(s):  
Viral Entry ◽  
High Titer ◽  
Cytoplasmic Tail ◽  
Cell Types ◽  
Spike Protein ◽  
Tangential Flow Filtration ◽  
Tangential Flow ◽  
Protein Functions ◽  
Flow Filtration ◽  
The Impact

The high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV) and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the protein’s function. Here, we optimized concentration methods for SARS-CoV-2 Spike pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers, but had no impact on sensitivity to convalescent serum inhibition. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but that this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions.

scholarly journals AAV Purification by anion-exchange chromatography

Bionatura ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Frank Camacho ◽  
R.P Cerro ◽  
N Varas ◽  
M.J Leiva ◽  
Jorge Roberto Toledo ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Large Scale ◽  
High Titer ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Raav Vector ◽  
Empty Capsid ◽  
Ratio Determination ◽  
Reproducible Method ◽  
Elution Step

We describe a new optimized, scalable and reproducible method based on anion exchange chromatography to obtain high titers of rAAV vectors without empty capsids contamination. The method takes advantage of Q-sepharose matriz to development a scalable procedure. After the virus harvest from supernatant and lysate cells, virus crude was subjected to anion exchange chromatography with Q-sepharose column. Three different protocols were tested, and the elution peaks were evaluated through qPCR rAAV titration and 260/280 nm ratio determination in order to identified empty capsid-containing fractions. A 150 mM NH4Ac wash step fallowing by 1 M NaCl elution step generate a a high titer eluted fraction of rAAV with 1.334 260/280 nm ratio. The described method makes rAAV vector purification an easily adapted for a large scale GMP production format to produce empty capsid free rAAV for clinics.

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