Importance and uses of albumin in serum-free eukaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell cultures
Albumin is a protein found in blood plasma and it is commonly used as a supplement to cell culture media. Albumins provide nitrogen and essential amino acids that are important for cell growth and help maintain osmotic balance in the cell culture environment. Additionally, albumins can act as a substrate for cell attachment, help to stabilize pH, and even protect cells from stress and damage. Albumin is often added to cell culture media at varying concentrations depending on cell type and the culture application.
Many molecules found in vitro are unstable or destructive when they exist in non-complexed forms. The primary function of albumin is to bind, sequester and stabilize a range of important small molecules and ions. In vitro, albumin acts as a multifaceted antioxidant. Its total antioxidant activity is a composite of many individual antioxidant activities. Albumin binds fatty acids and protects them from oxidation. It also binds copper, keeping it from participating in oxidation reactions. Albumin also binds cysteine, glutathione, bilirubin, and pyridoxal-5’-phosphate, protects the small molecules from oxidation, and is a sacrificial antioxidant.
Albumins are used in the bio-manufacture of therapeutic monoclonal antibodies and recombinant proteins. They are an important component of many serum-free cell culture systems such as those that utilize hybridoma or Chinese Hamster Ovary (CHO) cells. However, not all albumins have the same efficacy in culture media. Major factors that control the activity of albumin include the quality and relative quantity of specific ligands associated with the molecule. The ligands associated with albumin are determined largely by the nutritional status of the source animal and the purification process. This explains why the effectiveness of albumin(s) in a given cell culture system may vary and must be controlled. The ligand profile also helps to explain why natural albumin(s) derived from human (HSA) or bovine (FBS) sera perform differently than recombinant albumin(s) in cell culture.
Albumin can be incorporated into scaffolds to provide a source of essential nutrients and growth factors for cells, promoting cell survival and proliferation. Albumin also plays an important role in the field of tissue engineering, helping to improve the performance of tissue-engineered constructs and advancing the development of functional replacement tissues for medical applications.
Albumins are commonly used in biomanufacturing. They are used as a supplement in cell culture media to support the growth and health of cells used in the production of monoclonal antibodies, recombinant proteins, and other biologics. In addition, albumin can be used as a carrier for drugs, allowing them to circulate in the body for a longer period of time and enhancing their efficacy.
Biomanufacturers can achieve a more consistent, controlled, and reliable cell culture environment, reducing the risk of batch-to-batch variability and contamination associated with using animal-derived serum. This leads to improved product quality and consistency and helps to ensure the safety and efficacy of biologic products.
Overall, albumin is a valuable supplement in biomanufacturing, providing essential support for cell growth and health and contributing to the development of high-quality biologic products.
Some of the benefits of albumin supplementation are listed below. They help to demonstrate that albumin is among a small list of proteins that have profound value in cell culture.
Albumin is a highly soluble, 69 kDa, acidic protein. It can bind anionic, cationic, and neutral molecular species. Albumin has both high affinity and secondary binding sites for many molecules. Ligands bound to their primary site are typically non-reactive. Ligands bound to secondary sites are typically reactive.
At the functional level, albumin binds and delivers other molecules to cells in culture. The ability of albumin to support in vitro cell growth is largely determined by the type and quantity of nutrient ligands that it carries. A good appreciation of this can be gained by reviewing aspects of specific albumin ligand complexes.
Fatty acids, such as linoleic, linolenic, and oleic acid are insoluble in aqueous solutions and must be delivered to cells by a carrier molecule. Circulating albumin typically carries one or two free fatty acids. The binding of fatty acids also helps to stabilize the albumin. The activity of albumin as a cell culture supplement is partially dependent upon the specific fatty acids it binds and delivers to the cells.
Zinc and copper are present in serum. They are important to the health of cells and are required components of cell culture. A large proportion of zinc in serum is bound to albumin. Copper atoms can undergo univalent redox reactions and catalyze the formation of free radicals. This feature makes copper toxic to cells. In vivo, the potential toxicity of extracellular copper is mitigated when it is bound to albumin. There is one high affinity site for copper per albumin molecule. When copper is bound to this site, it does not participate in the redox reactions associated with free radicals. Albumin binds other divalent cations, such as Ca, Mg, Mn, Cd, Co, and Ni.
Human and bovine albumins contain an unpaired sulfhydryl at position 34 in their primary sequences. This sulfhydryl group often forms a covalent link with other sulfhydryl molecules such as cysteine or glutathione. Cysteine is not very stable in cell culture. It is easily oxidized to cystine and other oxidation products. By forming a protein mixed disulfide with cysteine and glutathione, HSA and BSA help to protect these molecules from oxidation and improve their availability for cells.
Pyridoxal, and its phosphate, pyridoxal-5’-phosphate, react non-enzymatically with amino acids to form Schiff bases. In aqueous solutions, especially in the presence of iron, these Schiff bases are unstable and result in the degradation of amino acids. Albumin binds pyridoxal at a site near its N-terminal. The binding of pyridoxal keeps it from reacting with and destroying amino acids in vitro.
Riboflavin can complex with tryptophan in aqueous solutions. In the presence of light, this complex decomposes into toxic products. Riboflavin and its phosphate, flavin monophosphate, are bound and protected from degradation by albumin. Albumin has a single binding site for tryptophan.
Many other molecules bind to albumin under physiological conditions. These include, but are not limited to anions, drugs, and hormones.
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