GFP: The Hello World of Biomanufacturing

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Submitted by Todd Smith on Thu June 27, 2019.

Beaker containing green fluorescent protein

Writing a program that displays or prints "Hello World" is a common way to demonstrate a computer language. It is so ubiquitous that "Hello World" has become a metaphor for any kind of demonstration system. For example, printing an ear is the "Hello World" of regenerative medicine. Green fluorescent protein (GFP) is easy to produce and its visibility in natural or ultraviolet (UV) light makes it easy to observe and follow through processes. Hence, purifying GFP is the "Hello World" of biomanufacturing. 

Fermentation room viewed from the BTEC lobby

As part of a recent professional development workshop on biotechnology, eight participants and I spent three weeks in North Carolina (NC) to learn about the state's biotech industry. We visited colleges and companies and gained hands-on experience with different aspects of biotechnology that emphasized analytical chemistry and biomanufacturing.

You might ask, with biotech so big and diverse, why focus on analytical chemistry and biomanufacturing? A quick inspection of NC biotechnology companies shows that many of NC's biotech companies have a pharmaceutical focus, thus it is not surprising that analytical chemistry and biomanufacturing would be an important aspect of NC biotech education.  Biomanufacturing is so important, that we spent a week at the BTEC (Biomanufacturing Training and Education Center). BTEC is hosted by North Carolina State University; Wake Technical Community College uses the facility for some of its biotechnology instruction.

The BTEC facility was designed to provide authentic opportunities for students to learn and practice skills in basic and advanced biomanufacturing. Its state-of-the-art equipment and instruments are also used to understand how the variables in different processes affect the outcomes. In our case, the week-long workshop at BTEC focused on making pharmaceutical grade GFP, that is, we had to complete our work as if we were in a regulated environment. We learned the differences between upstream and downstream processing. Upstream processing, while complex in its own right, is the step where bacteria or cells are grown. Fermentation is the process of growing bacteria. Cell culture is the process of growing cells.  Once the bacteria or cells are grown, the proteins or other products undergo downstream processing.  Downstream processing includes isolation, purification, fill and finish, and labeling.  

Our experiences, with pictures, are shared below. When we were not in the lab, we were receiving instruction on all the different aspects of the process. 

30 liter fermenter

Upstream Processing: During the first two days we grew E. coli containing GFP. The GFP gene is carried on a plasmid. The gene expression can be induced, causing the protein to be over expressed.  This process is similar to Genentech's method for human insulin production, which was licensed to Eli Lilly. 

In our class, we used 30-liter fermenters. Each fermenter has several input and output ports that are used to add nutrients and chemicals or obtain samples for process monitoring. On the first day, we familiarized ourselves with the parts and prepared the media and performed steaming in place (SIP).

On the morning of the second day, we inoculated the culture and monitored bacterial growth by following oxygen consumption, measuring A600 readings, pH, and glucose consumption. We learned that as oxygen levels dropped, the fermenter increases the amount of agitation, thus keeping the oxygen levels stable. At a critical point, we induced the expression of GFP by injecting IPTG (Isopropyl β-D-1-thiogalactopyranoside) into the fermenter. 

More photos are available at the Bio-Link Flickr site.

E. coli paste

Downstream processing: Step 1: Isolation (also called recovery). On the third day, we starting the isolation process. This involves harvesting the cells that have our product. GFP is not secreted into the media, hence the cells must be separated from the culture media by centrifugation. The photo shows the harvested E. coli under UV light demonstrating why GFP is the "Hello World" of biomanufacturing. Solutions containing high concentrations of GFP are green, and they fluoresce under UV light, which is cool and fun. 

Check out more isolation photos on our Flickr site.

Shearing device

After the cells are harvested by centrifugation, they must be disrupted to release the GFP. Disruption is accomplished, in this case, but pressure homogenization. A slurry of the E. coli cells is added to an instrument that "pushes" the liquid through a small orifice under high pressure. As the suspension clears this space, the pressure instantly drops and the cells burst open. 

Check out more isolation photos on our Flickr site.

GFP loading on the column

Once the GFP is "freed" from the cells, it must be purified away from all the other proteins, DNA, and biochemicals that are also released. We learned about the four primary chromatographic methods (size exclusion, ion exchange, reverse phase, and affinity) that are used for protein purification, on the third day.  On the fourth day, we used ion exchange chromatography to purify GFP. While there was enough GFP to see a faint green tint on the column, UV light showed GFP binding the column very well.  

More purification photos can be viewed on Flickr.

Paperwork
Did I mention paperwork? An important aspect of this class is understanding the process from a business perspective.  And, that involves running processes and operating equipment under regulated conditions. Each step must be fully documented including inputs, outputs, and observations. Steps and deviations must be signed, dated and countersigned and dated by lab partners. We also checked each others' calculations.
Getting ready

The last day was fill and finish. As is done in industry, biotech products that will be injected into animals– including people– must be packaged in highly sterile environments. Any bacterial or fungal contamination at this stage can be deadly to patients receiving the drugs. Highly sterile means suiting up, working in hoods, and not touching the product. We learned how to dress in protective clothing in ways that avoid touching the outsides of our garments. We also learned how to fill and manipulate vials using forceps instead of our hands. It was quite an experience. 

A virtual tour of fill and finish is provided on Flickr. 

Inspection

The last steps are the final inspection and labeling. As demonstrated in a recent court case* accurate labeling is critical. First, we inspected our product for any particulates. Next, we checked our labels and added them to the vials. Each team received 30 labels. After labels were applied the vials were counted and the number of remaining labels was documented. Before our product could be cleared, we had to have both the set of vials and number of labels recounted and signed off by a supervisor (in our case an instructor). If the numbers did not add up, an explanation would have to be documented, initialed, dated, counter initialed, and dated. Documentation is a serious matter. 

The virtual tour of fill and finish provided on Flickr also includes labeling. 

Final product
Once labeled, the product is ready to be shipped. In our case, that means we were able to take some vials of GFP home to show our friends and students. 

To review: Biomanufacturing involves upstream and downstream processing. In GFP production, upstream processing includes fermentation (or cell culture), and downstream processing encompasses the rest: isolation, purification, fill and finish, and labeling. Every step must be accurately documented and reviewed. 

Peering into a fermenter
Inside the centrifuge
Filling the homogenizer
Preparing the purification column
Collecting GFP
Preparing to fill vials
Documenting the process
Final product
Paperwork

* The court case, summarized in a Stat news article - "How a mishap at a photocopier derailed clinical trials and a development deal" - involved errors in a clinical trial where labels for a drug were accidentally switched, causing patients to receive half of the recommended dose. This accident caused the trial to fail and the responsible company was sued for $42M. Good systems of checks and counter-checks, as we learned, could have prevented the problem.