Degassing is the process of removing dissolved gases from a liquid. In FPLC, online degassing is achieved by passing the mobile phase through a degasser unit before it enters the column. The degasser unit uses vacuum pressure to remove dissolved gases such as oxygen and carbon dioxide, which can interfere with the protein purification process. This allows for the mobile phase to be free of any dissolved gases, resulting in improved protein yield and purity. Degassing the mobile phase prevents bubble formation. An online degasser is the preferred method.

One of the main benefits of online degassing is that it helps prevent protein denaturation. Denaturation is the process by which a protein loses its native structure and function. This can be caused by several factors, including changes in pH, temperature, and the presence of denaturants such as detergents or salts. However, dissolved gases can also cause denaturation by altering the pH of the mobile phase. When carbon dioxide dissolves in water, it forms carbonic acid, which can lower the pH of the mobile phase. This can cause proteins to become denatured, leading to reduced yield and purity.

In addition to preventing denaturation, online degassing also helps prevent protein aggregation. Aggregation is the process by which proteins form clumps (aggregates). Which can reduce the yield and purity of the protein sample. Dissolved gases can cause aggregation by altering the electrostatic interactions between protein molecules. For example, oxygen can react with sulfhydryl groups on proteins to form disulfide bonds, which can lead to protein aggregation. By removing dissolved gases, online degassing helps prevent these unwanted interactions and ensures a higher yield and purity of the protein sample.

Online degassing is also important for maintaining the accuracy and reproducibility of FPLC experiments. When dissolved gases are present in the mobile phase, they can cause fluctuations in the baseline of the chromatogram, making it difficult to accurately measure the absorbance of the protein sample. This can lead to inaccurate quantification of the protein yield and purity and can even result in errors in downstream applications such as protein structure determination. By removing dissolved gases, online degassing helps ensure that FPLC experiments are more accurate and reproducible.

Another benefit of online degassing is that it helps improve the stability of the protein sample. Dissolved gases can cause oxidative damage to proteins, leading to reduced stability and increased susceptibility to proteolysis. This can be particularly problematic for labile proteins or proteins that are sensitive to oxidative stress. By removing dissolved gases, online degassing helps protect the protein sample from oxidative damage, improving its stability and shelf life.

Online degassing is critically important for single pump gradient FPLC systems. In these systems the gradient is formed before the pump. Degassing before the valve inlets enables buffers to be bubble free. Bubbles in the valve inlet create inaccurate and irreproducible gradients.

Finally, online degassing can help reduce the cost and time associated with FPLC experiments. When dissolved gases are present in the mobile phase, they can cause column fouling, leading to a decrease in the efficiency of the chromatography. This can result in longer run times and increased use of mobile phase, which can be costly and time-consuming. By removing dissolved gases, online degassing helps prevent column fouling and ensures a more efficient and cost-effective FPLC experiment.

In conclusion, online degassing is an essential step in FPLC that helps prevent protein denaturation and aggregation, maintains the accuracy and reproducibility of experiments, improves the stability of the protein sample, and reduces the cost and time associated with FPLC experiments. By removing dissolved gases from the mobile phase, online degassing increases the reliability of the FPLC system.

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 When creating a vaccine, you need to attack the protein spikes on the SARS-CoV-2 virus. Proteins on the SARS-CoV-2 spikes have become the targets for creating vaccines and anti-viral monoclonal antibodies. Before analyzing the proteins of interest, they must be purified in solution. FPLC has been the gold standard for protein purifications for many years. Many prominent researchers create a multi-step purification protocol using FPLC resins and FPLC systems. ICON Scientific offers complete solutions, including a wide range of FPLC components that can be expertly configured to suit your research application. 

As the battle against SARS-CoV-2 virus continues to grow and enter new stages, researchers are relying on liquid chromatography techniques to provide accurate, efficient methods for analyzing potential vaccines and therapeutics.

Liquid chromatography (LC) is a critically important tool in therapeutic research, forming the basis of clinical trials based on RNA purification for the novel therapeutic vaccines. HPLC has been instrumental in pioneering research into the composition, method of action and structure of SARS-CoV-2. High Performance Liquid Chromatography (HPLC) is critical for quality control and quality assurance for the bioactive ingredients used in vaccines as well as therapeutics and all aspects of pharmaceutical development.

Liquid chromatography is a vital technique in many industries including pharmaceutical, production, MRNA production and development, cannabis/hemp analysis, food and beverage industries, manufacturing, quality control and more.

Vaccines need to be purified and filtered at different stages. One of the most important purification steps involves separation techniques using chromatography columns. In liquid chromatography the dissolved liquid (mobile phase) carries the analyte of interest through the column which is packed with a fixed material (stationary phase). Different chemical properties of the molecules within the analyte determine the retention time for the components of interest.

Liquid chromatography (LC) is an essential technique used for the purification of biomolecules including RNA. SARS-CoV-2 is an RNA-based virus. RNA purification uses liquid chromatography (LC) because it is performed at both higher temperature and higher pressure than normal LC. Higher pressure and higher temperature stimulate the denaturation process to remove protein complexes. The combination of high pressure and temperature and a binary solvent gradient prevent RNA degradation, while providing an effective RNA isolation. Liquid chromatography instruments can isolate the targeted messenger RNA, the mRNA and therefore analyze the vaccines efficacy.

Preparative HPLC pumps are used in the manufacturing of vaccines. Liquid nanoparticle (LNP) production equipment is used for the development of mRNA-based vaccines, lipid nanoparticles (LNPs) have proven to be a suitable delivery form. Lipid nanoparticles protect and encapsulating the active ingredient allowing it to be transported to the target cells in the human body. SARS-CoV-2 vaccines are the first large-scale production vaccines using lipid nanoparticles and mRNA in vaccine history. Icon Scientific supplies custom solutions for both small and large-scale production of vaccine lipid nanoparticles.

SARS-CoV-2 researchers are constantly fighting a battle as they screen potential antiviral treatments and continue to perfect vaccine development. 

During these unprecedented times ICON Scientific can help you configure and customize liquid chromatography (HPLC) systems to assist your research into the structure of SARS-CoV-2 and its method of action.

Any Questions? Call us at 301-330-4266

Or send us an email at Icohen@iconsci.com

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