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Cesium Chloride Density Gradient Ultracentrifugation Workflow

Solutions for every step

of your AAV workflow

 

CESIUM CHLORIDE DENSITY GRADIENT ULTRACENTRIFUGATION

 

Get all the AAV reagents you need from one trusted source. Upstream. Downstream.

And the downstream of the upstream process too. Using a different downstream processing method? 

We also have dedictated worfklows for Chromatography and Iodixanol Gradient Ultracentrifugation.

 

Cell Expansion and Transfection

We understand the importance of your upstream conditions and the critical nature of optimizing both the cell media and transfection reagent utilized. Our team of scientists, process engineers, manufacturing specialists, and quality control experts are here to help define and test custom reagents for your AAV needs.

 

Get in touch with our team to discuss the best solution for your specific application.

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See our findings on optimized formulations

Explore our research to learn more about how we can optimize our custom formulations for your cell expansion and transfection needs.

Cell Lysis and Endonuclease Treatment

Efficient cell lysis and endonuclease treatment are essential for maintaining high AAV yields and purity. We have proven cell lysis buffers in stock in multiple sizes to match your bioreactor needs, as well as Benzonase® Nuclease to reduce product related impurities such as residual host-cell DNA and plasmid.

 

Our Mammalian Cell Lysis Buffer is a polysorbate 20-based cell lysis reagent at a 10X concentration. The 100 mL volume is sufficient for 1 L of cell culture harvest, and the 500 mL volume is sufficient for 5 L of cell culture harvest.

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Precipitation

Following lysis, endonuclease treatment, and the clarification of the culture harvest, precipitation can be used to reduce impurities, including proteins and nucleic acids, and to concentrate the sample prior to ultracentrifugation.

 

PEG-8000 with Sodium Chloride (NaCl) is ideal if you are using a single precipitation step method. For a two precipitation step method, you can use Calcium Chloride as a precipitant, followed by PEG-8000 with NaCl. We have off-the-shelf stocks of 1 M Calcium, 40% PEG-8000, and 2.5 M NaCl in stock and ready to ship.

Filtration buffer
Ultracentrifugation

Cesium Chloride (CsCl) forms a linear density gradient under ultracentrifugation that is used to separate empty from full AAV capsids into distinct bands.

 

There are multiple approaches to prepare the CsCl gradient for AAV purification:

 

  • Directly mixing the AAV sample with a saturated solution (1.9 g/mL).
  • Overlaying the AAV sample over a single CsCl solution (1.4 g/mL).
  • Using a double layer approach with two different CsCl concentrations (1.5 and 1.3 g/mL).
Filtration buffer
Dialysis

Using dialysis, the CsCl-containing buffer will exchange with the dialysate to a more suitable solution for either storage or continued preparation. We have multiple options to perform dialysis using your buffer solution of choice, including 1X PBS with 5% Sorbitol or 50 mM HEPES (pH 7.4), 0.15 M NaCl, and 25 mM EDTA (HNE) buffer.

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Storage

We have extensive experience manufacturing custom storage buffers. Our team of scientists, process engineers, manufacturing specialists, and quality control experts are here to help define custom storage reagents to meet your needs.

Fill out our custom order form or get in touch with our team to discuss the best solution for your specific application.

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Fully customizable

Need that in a 1 L bottle filled to 500 mL?
Or at a different pH for your DOE?

From formulation to format, quality control testing, documentation, and labeling, we can manufacture exactly what you need. Quickly tailor small volumes of buffers for your early-stage research with our Build•Tek™ Custom Configurator, or consult with our experts to find the best solution for your specific application.

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Our AAV experts are here to help every step of the way

Our scientists, process engineers, manufacturing specialists, and quality control experts are here to help. We can help you find the ideal solution for your specific application.

R&D team

Find out what’s possible

 

Talk to our consultants today to discover custom solutions that can help you achieve your goals.

 

Get in touch:

1.800.209.4488

Monday to Friday

8:00AM–5:00PM PT

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