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.

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Minimizing Cleaning Validation

Preparing for large scale plasmid purification.

One of the major challenges of plasmid manufacturing is cleaning validation. To avoid excess costs and time, disposable technology can decrease validation needs for both the cleaning and sterilization processes of fixed equipment systems.

Disposable materials for use in pharmaceutical manufacturing have come a long way from traditional reusable technology. From pipettes and syringes to disposable bioreactors and pre-packed purification columns, disposable single-use materials have many advantages for pharmaceutical manufacturing, including:

  • Low startup cost
  • Pre-sterilized components and assemblies
  • Plug-and-play operations
  • Increased flexibility in design and operation of systems
  • Reduced validation time and cost
  • Reduced changeover time

These advantages have stimulated the rapid adoption of disposable technology in clinical and small scale production applications. Single-use disposables, today, include flexible tubing systems with either direct aseptic connections or tubing fusing systems, process mixing containers, large scale bioprocess reactors, and sensors for the measurement of process parameters.

The advances in single-use disposable materials are making it easier for the Contract Manufacturer to meet the diverse and exacting requirements of researchers and gene based therapy companies in need of additional processing capabilities.

-Jerry B.

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From Dreadful to Enjoyable: Transform Your GMP Training

Given the right setting, GMP training can be fun and interesting.

Most companies struggle with how to make GMP training more fun and exciting. As the Quality Assurance director of a company that provides contract plasmid manufacturing, it seems almost impossible for me to find time to be creative.

This year for the “dreaded” GMP training we played a game – yes, a game – called Wheel of Compliance.  This training was a “spin-off” of Wheel of Fortune.  We had a wheel and everything!  A very colorful one I might add.

The employees were split up into 3 teams, and, one by one, a player from each team spun the tri-colored wheel.  The color the ticker landed on determined the difficulty of the question.  To encourage learning and participation, points were only awarded for CORRECT answers and prizes were given to the winning team.

Trust me, games bring out competitive spirit and, therefore, are very effective learning tools.  Everyone listens to ALL the questions to test their own knowledge.  It is an interactive, fun way to learn!

As an annual tradition, we invent a new game for GMP Training Day.  In 2008, we played GMP Baseball.  It was Team Plasmids vs. the DNA Vaccines. Participants actually ran the bases for correct answers.  Questions corresponded to the types of hits and were based on difficulty level (i.e. the question related to a single is easier than the question for a homerun).  In 2009, we played Jeopardy.  Both regular and double Jeopardy rounds were included as well as a Final Jeopardy question!

So, get out there and create innovative training sessions!

The usual groans and yawns that accompany the formidable training day will soon be transformed into laughs and competitive spirit.

If you have any great ideas, please share them in the comment section below!

– Dorothy P.

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