BTEC Day 3: Pressure Does the Trick

The third day of the biomanufacturing bootcamp was dedicated to cell disruption and centrifugation. In smaller scale conditions these are processes that would normally not take an entire day, but we had a lot of bacteria that needed to be busted open. After all, that is where our green fluorescent protein (GFP) is!

GEA Niro soavi uses 21K psi to disrupt cells.

The GEA Niro Soavi homogenizer disrupts cells by generating incredible pressure and forcing the slurry through a small hole. When the cells come out the other side, the drop in pressure is so great that the cells explode. To ensure complete homogenization we ran the batch through the machine twice. Our homogenate (i.e., all the busted open bacteria) contains the precious GFP, but we need to separate it from all the other bacterial junk.

So, centrifugation was next. We used a disc stack centrifuge, which is a remarkable piece of engineering. Inside the conical dome there is a series of stacked discs that separate the liquid containing the protein of interest and the solids which are cellular debris. The disc stack has a large capacity so it can be continuously fed with homogenate. The exiting fluid is given a little back pressure to ensure no debris makes it through into the liquid product (supernatant).

A disc stack centrifuge is continuously fed.

Once processing was complete, the machine needed to be taken apart and cleaned. Inside the dome of the centrifuge we saw one of the end products of our homogenization and centrifugation steps — the leftover paste that represented the cellular guts of trillions of bacteria. We started with approximately 13 liters of bacteria liquid culture from the bioreactor and reduced that to enough cellular debris to fill a small ziplock sandwich bag. I was curious to see how much GFP was still in the paste (or pellet) that would be discarded so I ran a black light over it. It glowed green under the light so we know that we had some product loss at that step.

Cellular debris from 13 liters of bacteria culture.

Now we have officially left upstream manufacturing and are ready to begin purifying our product. Almost every step has been very close to an industry process up to this point. However, It would be difficult for us to purify this quantity and still finish the class this week so we need to work with smaller volumes. The next step will involve strong anion-exchange chromatography to purify the GFP. We will need to dilute the supernatant we collected from today to reduce conductivity so our GFP will bind to the column. We will also need to adjust the pH and filter the supernatant prior to the chromatography step.

One thing I learned today was that no matter how new or sophisticated the equipment there are always peculiarities to get used to. For example, all the spinning parts on our centrifuge were reverse threaded to avoid the problem of the centrifuge loosening the fittings and screws during operation (which could be disastrous considering it spins incredibly fast). So, reverse threading makes sense from an engineering perspective but it is easy for newbies to forget while you are assembling or taking apart the centrifuge. We were warned several times about “galling” the stainless steel fasteners by going the wrong way. It was a little nerve-racking because some parts would cost over $4000 to replace!

July 11th, 2013 | Category: Announcements

“Once a fermentation has started it can only get worse, not better.” The quote was attributed to DJ Hockenhull, who used fermentation to produce penicillin in the 1940’s. Today, bootcamp was all about fermentation. To start off we added glucose and ampicillin to prepare the media for fermentation with E. coli. The strain we are using is transformed with a plasmid containing the lac operon and the gene for green fluorescent protein (GFP). One difference from the lab I teach on GFP is that the inducer is IPTG, not arabinose, but otherwise everything is very similar.

Bioreactor bubbles with 20 liters of bacteria.

We inoculated the media with our bacteria through the bioreactor’s feed valve. At that point the fermentation was officially in the lag phase. It is typical to use 3-10% of the final volume for your inoculation volume. We were closer to 10% so we would quickly move out of the lag phase and into the log growth phase. The bioreactor is equipped to control temperature, agitation speed, dissolved oxygen, pH, pressure, and airflow. We could also have added an anti-foaming agent into one of the ports but it was not necessary for this process. In addition, we sampled our batch frequently through the sample valve to monitor the progress of the fermentation. We measured OD 600 using a spectrophotometer and glucose concentration using a bioanalyzer (YSI 2700 Select). The whole run took about six hours and then we harvested our cells and cleaned the bioreactor. All that was needed after that was to wash the cells to prepare them for the next day: cell disruption and centrifugation.

Glowing green fluorescent protein.

If there was one pull quote from today’s class, it would be that “the bioreactor is the queen” of the fermentation process. Everything in upstream processing should ultimately optimize the use of the bioreactor for fermentation — in other words, everything should serve the queen. If the queen isn’t happy, no one should be happy because it means that money is being lost. Oh, and don’t let the smell of fermentation put you off. That’s the smell of money!

July 10th, 2013 | Category: Announcements

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.

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 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.

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!

July 8th, 2013 | Category: Announcements

The California Community Colleges Chancellor’s Office has released comparative salary data for graduates of the California Community Colleges system on a website called Salary Surfer. Not surprisingly, salary rises dramatically after completing a certificate program or two-year degree, regardless of the discipline of study. Technical disciplines like industrial technology, engineering, and allied health have the highest pay after graduation. Biotechnology (including biomanufacturing) graduates also do well:

Note that after two years of employment, biotech A.S. degree graduates earn much higher wages than general biology graduates, and biotech certificate earners do nearly as well as general biology grads. After five years of employment, the median biotech A.S. degree graduate wage is $55,673 – good news for all those pursuing biotech or biomanufacturing degrees right now!

If you expand the degree categories, the site shows community colleges that have programs in that category. For instance, expanding the “Biotechnology and Biomedical Technology” category reveals this:

Laney College has programs in biomanufacturing and medical device manufacturing. As you will discover on Salary Surfer, a potential biotech student has many options to choose from when selecting a college, especially in the Bay Area.

June 21st, 2013 | Category: Announcements

I have recently become involved in a non-profit genomics company, Profile Genomics, located in Alameda next to an Estuary Crossing Shuttle stop and College of Alameda. Profile Genomics is the business side of the Merritt Genomics program and is currently doing contract research work for hospitals and research labs. What is so exciting about this company is that students do the lab work and gain valuable experience while completing their educational programs. It helps address the common problem of community college students completing certificate or A.S. degree programs in biotechnology but not being able to land a job due to lack of work experience.

The contract organization idea is not new. CXO is a general term that includes Contract Research Organization (CRO), Contract Manufacturing Organization (CMO), Contract Testing Organization (CTO), and other iterations. CXOs are an innovative way for community colleges to offer students  work experience while in their biotech programs. Nationwide there are several examples of successful CXOs*. Salt Lake Community College has two CXOs called InnovaBio and STUDENTfacturED. Pasadena City College went with a CXO incubator model called the Pasadena Bioscience Collaborative. CXOs are a true win-win for everyone involved. Students get paid as employees and gain work experience, college programs gain an income stream to invest in their labs and expand course offerings, and companies get projects completed that would otherwise be dropped due to cost or time concerns.

So, if you are interested in gaining experience and working with the latest in genomics technology, check out Profile Genomics and the Merritt Genomics program. Go to their websites to find out about upcoming classes and open lab times.

*Source: Bio-Link Summit Report on Contract Organizations

May 23rd, 2013 | Category: Announcements

The word theory is used often in daily language. You may have heard it used like this: “My theory is that she secretly likes him” or “My theory is that they really just wanted to hire from the inside” or even “There are a number of different theories about the cause of the disease.” So it’s understandable why so many people think that theories are sort of off-the-cuff, plausible explanations made with little to no evidence. In science we mean something entirely different when we talk about theories, however. Let’s look at two definitions of the word.

From the Oxford American Dictionary:
theory |ˈθēərē; ˈθi(ə)rē|noun ( pl. -ries)a supposition or a system of ideas intended to explain something, esp. one based on general principles independent of the thing to be explained : Darwin’s theory of evolution.

From The Essentials of Biology with Physiology (standard non-majors biology textbook):
theory: a widely accepted explanatory idea that is broad in scope and supported by a large body of evidence.

So, theories in the scientific sense of the word are broad ideas with great explanatory power. This is in contrast to another sciencey word that we use a lot less in everyday conversation – hypothesis. A hypothesis is a tentative explanation that a scientist proposes for a specific phenomenon that has been observed. The biology textbook referenced above calls it “an idea on trial” and that’s a fine shorthand way of thinking about it. In fact, it is hypothesis that we mean when we use the word “theory” in conversation. Hypotheses are more conjectural by nature because they are backed up by less evidence than theories.  It would be more accurate, but perhaps more geeky, to say the following: “My hypothesis is that I got my cold from my roommate.”

Theories are powerful ideas that allow us to learn new things by spinning off multiple new hypotheses. It’s sort of like a large, successful company spinning off smaller companies. Indeed, scientific theories have led to some major breakthroughs in technology and huge profits by companies. Let’s take Einstein’s theory of relativity. One of the key parts of Einstein’s theory, called special relativity, deals with time dilation, the idea that a moving clock will tick-tock more slowly than the observer’s stationary clock. It’s a strange idea that time would work differently depending on how fast things are moving and where you are relative to another moving clock, but it has huge implications for satellite communications and global positioning systems (GPS). The other idea covered in Einstein’s theory of relativity, general relativity, has to do with gravitational effects on time. Both movement and gravity have to be accounted for in order to make the satellites work properly. It all gets complicated very quickly so I’ll let this video do the explaining (from the Science 2010 Visualization Challenge).

Pretty fascinating stuff, huh? The theory of relativity is so powerful that we can use it to ask new questions and propose ever more hypotheses. For instance, does the fact that your head is higher off the ground than your toes mean that your hair ages faster than your toenails? If the theory of relativity is true, we would hypothesize that this is the case. After all, doesn’t time move faster the further away you are from a major source of gravity? We would expect that the time difference would be a vanishingly small number and it would require a super-sensitive clock to measure it. In fact, this experiment has been performed using the most sensitive clock known, the optical clock, which is 40 times more sensitive than the best atomic clock. Result? Yes, the hair on your head ages faster than the nails on your toes…by 4-parts-in-100-million-billion, a number so small it really wouldn’t be worth your while spending as much time as possible laying down flat in order to keep your brain from aging too fast.

Theories don’t age or wear out with time. The theory of relativity was proposed between 1905 (special relativity) and 1916 (general relativity). Of course, it was a fundamental property of the universe before Einstein discovered it and it has been ever since. Further experiments have only added more evidence supporting the theory of relativity, including some recent work by Stanford scientists. There is such massive evidence supporting the theory of relativity that it is a scientific “fact” in much the same way the heliocentric theory of the solar system, atomic theory, cell theory, gravitational theory, plate tectonics theory, and evolutionary theory are facts.

There’s much more to say about evolutionary theory, which is why I treat it as a major theme of my general biology classes. If you’re interested in finding out more about the evidence, Berkeley’s Understanding Evolution website is an excellent resource.

Image from here.

June 20th, 2011 | Category: Science news