Recent breakthroughs in COVID-19 treatment have put mRNA vaccines and technologies in the spotlight. Cold chain management is a crucial part of the mRNA production and distribution process.Temperature also plays a vital role in the laboratory production of mRNA, specifically within the enzymatic reactions and purification processes inside the bioreactor.
This App Note provides:
- A step-by-step overview of mRNA production and distribution
- Insight into the importance of cold chain management
- Consideration of vaccine shipping and storage challenges
- Insights into current biotechnology & biopharma trends
- Several solutions for bioreactor temperature control
Many researchers claim the mRNA vaccines are just the beginning, and the production of mRNA treatments will change the standard of care and usher in a powerful era of personalized medicine. While mRNA therapies hold a lot of promise, there's still much for us to learn about the best way to manufacture these nucleotide-based drugs.
Download this app note to learn how effective temperature control management can help manage workflows and increase yields and throughputs for mRNA.
JULABO USA_App Note_mRNA
APPLICATION NOTES
Bioreactor Temperature Control for the Production of mRNA
Recent breakthroughs in COVID-19 treatment have put mRNA vaccines and technologies in the spotlight. Many researchers claim the mRNA vaccines are just the beginning, and the production of mRNA treatments will change the standard of care and usher in a powerful era of personalized medicine. While mRNA therapies hold a lot of promise, there's still much for us to learn about the best way to manufacture these nucleotide-based drugs.
Cold chain management is a crucial part of the mRNA production and distribution process. In the case of the COVID-19 vaccines, shipping and storage challenges became a hot topic. While you may not hear as much about it, temperature also plays a vital role in the laboratory production of mRNA, speci?cally within the enzymatic reactions and puri?cation processes inside the bioreactor. In this application note, we'll explore manufacturing and temperature control considerations related to mRNA production and how effective temperature control management can help manage work?ows and increase yields and throughputs for mRNA.
Ph.D.
resid
by Dr. Dirk Frese
VP of Sales, Marketing & Service, JULABO USA
An Overview
of mRNA Production
Unlike producing traditional pharmaceuticals or working with small molecules and biologics, the best process for producing mRNA hasn’t been de?ned or standardized. Many of those working in the production of mRNA have their own, proprietary approach. For the sake of this overview, we’ll outline an example for producing mRNA for the SARS-CoV-2 vaccine, this is one approach among many that may be used to create viable mRNA.
Note: The steps and process outlined below are for example only, there are many production routes that you can take, and you have to decide what’s best for your facility and application.
1. Create DNA Duplicate DNA
2. Convert DNA to mRNA
3. Encapsulate mRNA into nanoliposomes
4. Test, Package & Ship
NOTE
Bioreactor Temerature Control for the Production of mRNA
Create DNA
Step 1
JULABO
Synthesize a short segment of genetic material/ DNA for a speci?c protein such as the SARS-CoV-2 spike protein.
Circulators
Duplicate DNA
Note: A variety of temperature control equipment is necessary to facilitate the processes inside the bioreactor, including laboratory circulators, such as JULABO USA's FL Series, SemiChills, PRESTO, DYNEO, and MAGIOs. JULABO also offers water baths for the DNA cloning process.
Insert the DNA sequence in a bacterial plasmid (pDNA) using restriction endonucleases (enzymes). This process is called cloning. It can be done in a water bath or similar at 37°C. The resulting engineered plasmid enters into the bacterium (E.coli) by means of a bacteriophage (transfection).
Maintain
Culture the E.coli in an Erlenmeyer ?ask. Add nutrients such as sugar and nitrogen, sources and salts, which the E.coli love; shake it up to get enough oxygen so that it multiplies. It's essential to maintain the temperature at 37°C (same as the human body temperature) during this process. The bacteria multiply quickly every 20 minutes also producing more plasmid DNA material.
37°C
Put the contents from the Erlenmeyer ?ask into a bioreactor/fermenter, this is usually a jacketed steel or glass reactor. A circulator, such as those offered by JULABO USA, keeps the temperature consistent in the bioreactor by circulating water or thermal ?uids through the reactor jacket to maintain the required 37°C. The circulator constantly removes the heat caused by the bioreaction (metabolism) and counter-cools the bioreactor. When it comes to temperature, there’s very little room for error as E.Coli will die at 42°C.
Harvest the E.coli cells by centrifugation of ?ltration. Cool to 0°C.
NOTE
Bioreactor Temerature Control for the Production of mRNA
Step 1 (continued...)
Open cells with SDS detergent and denature DNA material by adding ethanol or isopropyl alcohol, add potassium hydroxide or sodium hydroxide so that the DNA forms clumps, maintaining the ice-cold, 0°C temperature.
Remove excess material, including chromosomal DNA material, through centrifugation/chromatography columns. This continuous process ?lters the DNA through columns to ?lter out the plasmid, wash out hydroxides and neutralize/renature, and remove cell debris. JULABO units are also helpful in cooling the chromatography column to maintain adequate temperatures.
Cut the circular plasmid molecule, open it up using enzymatic scissors and incubate the mixture again at 37°C in a bioreactor. Again, you'll need a precise temperature controller/circulator to maintain the vital temperature.
Purify DNA through chromatography and pull apart (melt) the double helix DNA with heat at 50-100°C. A heating circulator or refrigerated/heating circulator helps facilitate this process and maintain proper temperatures.
Step 2
Convert DNA to mRNA
An enzyme, RNA polymerase, is added to transcribe mRNA molecules from the DNA templates. This essentially creates a desirable mRNA for further use. Incubate at 37°C in the bioreactor using a laboratory circulator to maintain the temperature.
Chill
Further capping and polishing steps may happen depending on the speci?c protocol. These are all enzymatic steps in small bioreactors.
-40°C
Chill the mRNA to -40°C, then -70°C. A recirculating chiller with a working temperature down to
-70°C or below is necessary for this step. The next step for some labs may be transforming the chilled mRNA to other locations, including in some cases, out of the country or out of state.
to
-70°C
then
NOTE
Bioreactor Temerature Control for the Production of mRNA
Step 3
Encapsulate mRNA into Nanoliposomes
We can't inject mRNA directly into the body as it would degrade instantaneously, so we add fats, lipid particles, and polyethylene glycol to the mRNA mixture to form droplets of mRNA encapsulated in the fatty acid, the so-called liposomes.
Then we have to remove the ethanol and deep freeze, typically done through a centrifuge. Ethanol will collect as the supernatant on top of the aqueous liquid.
Step 4
Test, Package & Ship
Additional cold chain management is applied to maintain the temperature consistency throughout the ?nal steps, including packaging, shipping, and delivery. This may vary from vaccine to vaccine between -20°C to -70°C.
Application Challenges & Work?ow Automation
Because the process for manufacturing mRNA is relatively new, production standards have yet to be de?ned, and pharma and biopharma companies may ?nd themselves wondering how to scale production for higher throughputs. Regardless of the process or approach, temperature control is a vital part of scaling the process, and the temperature control unit plays a key role in helping manage, capture, and track data for larger bioreactors.
Additionally, some laboratories are already exploring how to optimize the mRNA production process to decrease bottlenecks, reduce costs, and address the shortage of raw materials, including the availability of speci?c enzymes and nucleotides. More biotechnology and biopharma companies are looking at ways to manufacture mRNA by mimicking nature, including the use of glucose, water, and microbes rather than enzymes and nucleotides. Other labs are looking at ways to produce nanoliposomes in-house. As mRNA production and processing evolve, bioreactors and bioreactor temperature control will continue to play an essential role, making the selection of a temperature control unit for the bioreactor vital to future success.
NOTE
Bioreactor Temerature Control for the Production of mRNA
Bioreactor Temperature Control Considerations
When selecting a temperature control unit for a bioreactor, you’ll want to be sure you’re getting a unit that will improve your processes, protect your reactor, and meet your objectives. Whether you’re looking to scale your production, start your exploration into mRNA processing, or maintain your throughputs with greater ease and accuracy, the right temperature control unit is necessary.
Here are a few questions to consider:
How large is the bioreactor vessel you’ll be using in terms of volume?
Which temperatures are you trying to reach and maintain?
Are there any speci?c interfaces required? Will you need to integrate with your PLC? How much data capture and control do you need?
How much space do you have in the lab, and how close should the circulator be to the bioreactor?
Do you need password protection or the ability to program and save steps?
Are you operating under GMP or cGMP practices?
1-1000
Liters
Do you need a temperature control unit with IQ/OQ documentation?
Bioreactor Temperature Control Products & Recommendations
JULABO USA offers a range of benchtop units to operate bioreactor vessels from 1 liter in size to 20 liters, with additional units to heat and cool reactors up to 1,000 liters. Our team is happy to help you size and select the circulator or temperature control unit that’s best for your lab.
All of the JULABO USA circulators have RS232 communication and other I/O capabilities for integration into PLC systems for automated control and data capture. We can also execute or help you accomplish any IQ/OQ requirements for GMP and cGMP compliance. Depending on the size of the vessel, the amount of automation you need, and the features and bene?ts that matter to you, we can help you ?nd the right temperature control unit for your mRNA and mRNA-related processes.
Visit www.JULABO.us for more recommendations!
FL Series SemiChill PRESTO DYNEO DD MAGIO
NOTE
Bioreactor Temerature Control for the Production of mRNA
Conclusion
Regardless of where you are in your mRNA research, discovery, or production process, we’re happy to discuss your bioreactor temperature control options. Circulators are only part of the overall temperature control management chain, yet it’s a critically important part. Before you go it alone, talk to our team. We’re happy to provide personalized product recommendations based on your bioreactions and your goals.
sales@julabo.us (610) 231-0250
Explore Our Products & Services
Download Our App Now!