AIRMIX – A Softer, Gentler Lysis

large scale gmp plasmid manufacturing

E. coli cell paste.

Arguably, the most challenging step in plasmid manufacturing is cell lysis. Thousands of times larger than a typical small molecule pharmaceutical API, a plasmid is a large circular molecule that most of our clients prefer in the supercoiled form.

Several things can go wrong if the lysis step is too harsh. First, the plasmid molecule will shear or unwind to form an open circle. Second, the host cell’s genomic DNA will shear making it difficult to separate from the plasmid.

Knowing this, VGXI spent the first 2-3 years of operations developing its patented lysis process. The process had to be gentle enough to not shear the plasmid or genomic DNA but stringent enough to break open the cells and separate most of the cellular debris and genomic DNA from the plasmid. Using conventional methods (manual lysis by hand), a 50 kg batch of cell paste would have taken 3-4 months to lyse. Our goal was to produce an automated process that could lyse 50 kg of cells in less than a day.

The process we developed employs tanks connected in series that perform different mixing and filtration steps. There are a couple of key mixers in the machine, such as our patented “bubble column” mixer. This is a long tube full of bubbles that the cell lysate runs through. The bubbles gently separate the lysed cellular components without shearing the plasmid or genomic DNA. As the cell solution runs through the bubble column, special chemicals in the lysis solution cause the genomic DNA to tangle with the cellular debris. The cell debris and genomic DNA then float to the top of the lysis solution and form a thick, oatmeal-like cake. The solution containing the plasmid is pulled off the bottom of the tank for further processing.

Where it used to take 2-3 days to lyse a large scale batch, it now takes only a few hours. Furthermore, VGXI’s patented bubble mixer removes over 99% of cellular and genomic DNA contaminates simplifying downstream purification.

-Henry H.

Advertisements
This entry was posted in Uncategorized. Bookmark the permalink.

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s