Laney College

BTEC Day 1: “Dead Legs” are Deadly

BTEC Day 1: “Dead Legs” are Deadly

Today was the first day of the Biomanufacturing Bootcamp at the Golden LEAF Biomanufacturing Training and Education Center (BTEC) at North Carolina State University (NCSU). The facility is located on the Centennial Campus of NC State and has 82,500 square feet of laboratory and classroom space, including a simulated commercial cGMP production facility.

Front entrance of BTEC with bioswale in foreground.

Front entrance of BTEC with bioswale in foreground.

Our session began with a review lecture about FDA regulations (CFR 210 and 211), drugs versus biologics, ICH Q7, documentation, and quality. We also reviewed the biology of production of a recombinant DNA product. This week we are using E. coli to produce green fluorescent protein (GFP), a common exercise in biotech programs across the country including the biomanufacturing program at Laney College. The key difference is scale. I am used to 3 to 5-liter tabletop bioreactors. These glass bioreactors are relatively easy to set up and operate. At BTEC we are using a 30-liter stainless steel bioreactor with ports, valves, condensers, inlets, outlets, and gauges all over the place. Our first activity in the lab was called “Training- Fermentor component identification” and it took quite a while for t

hree teammates to find 28 critical parts on the bioreactor. The good news is that these bioreactors are designed for research and pilot plants so they have many more ports and fittings than a bioreactor would have in a full production facility.

Sartorius 40-L fermentation vessel and controller.

Sartorius 30-L fermentation vessel and controller.

After another lecture we were ready to set up our production run. We used batch production records (BPRs) for our work instructions and documentation. Our three procedures were pressure hold, media preparation, and media sterilization. In pressure hold we tested that the bioreactor vessel and all the valves and ports had tight seals. Any leak could introduce contamination into the process. Our BPR had an area for the operator to sign and date and record any data and an area for verification signatures and dates by teammates. During the pressure hold test, we discovered that one bioreactor was having trouble holding pressure so the lab technicians and students had to figure out the source of the leak. Media preparation was fairly straightforward. Bioreactors of this size have load cells which weigh the bioreactor as you add batch ingredients to the vessel and internal impellers to mix the solution. As we added water and broth solutes to the vessel we monitored the vessel’s weight to ensure we were adding the right amounts.


Nothing like a sterilized bioreactor to brighten your day.

Proper sterilization is key to fermentation. We learned in lecture that the many pipes, valves, ports, and so on can have “dead legs,” which means they can be difficult to sterilize in place (SIP). Dead legs should be minimized as much as possible when designing a bioreactor. Bioreactors are often supplied with several steam sources for sterilization. For instance, SIP needs to be done every time a sample is taken from the sample port. So, David Yarley, our main bootcamp instructor, gave us the memorable phrase “dead legs are deadly” to drive home this point. Dead legs are potentially deadly to fermentation because a contaminant from them can cause a complete loss of a batch.

After a hard day's work.

After a hard day’s work.

Our first day ended with our media prepared for inoculation (or “seeding”) the following morning. The entire class of 13 students, all faculty from various colleges and universities from around the country, decided to celebrate the successful setup of our bioreactors by enjoying some North Carolina barbecue. Barbecue is serious business in North Carolina, with Eastern and Western styles both vying for the title of the authentic state barbecue. The battle has been so heated that the state legislature has gotten involved. All I will say on the matter is that there is nothing like it in California!