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  Albumin in Cell Culture
Importance and uses of albumin in serum-free eucaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell cultures
 

Table of Contents

Albumin, a Serum-Free Media Supplement, Useful In Biomanufacturing, Tissue Engineering and Specialty Media:
Albumins are used in the biomanufacture 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. 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.

Possible contamination of HSA or FBS sourced proteins such as albumin with adventitious agents is a concern to many biomanufacturers. The remote potential contamination of bovine albumin with Bovine Spongiform Encephalopathy (BSE) virus has prompted many companies to try to avoid the use of albumin, and other animal-source proteins. This has led to the emergence of animal-component free (ACF) media.  This is a trade-off. The performance benefits of using albumin in culture media should be carefully weighed against the risks of contamination.

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. For a more complete discussion of albumin as a cell culture media supplement and its associated ligands go to Sigma's Media Expert.

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Primary Functions of Albumin in Cell Culture Systems:
Many molecules found in vitro are unstable or destructive when they exist in non-complexed forms. A 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. It's total antioxidant activity is a composite of many individual antioxidant activities. Albumin binds fatty acids and protects them from oxidation; binds copper and keeps it from participating in oxidation reactions; binds cysteine and glutathione and protects them from oxidation, binds bilirubin and pyridoxal-5’-phosphate and protects them from oxidation, and is a sacrificial antioxidant.

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Chemical Attributes of Albumin that make it a Useful Serum-Free Media Supplement:
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.

Amino Acid Sequences of Human and Bovine Albumin Precursors: The following are generally accepted sequences, however variants and mutations also exist. Bovine serum albumin preprotein (607) is two amino acids shorter than human serum albumin preprotein (609). The first 18 amino acids of albumin precursors provide secretion signals; the next 6 amino acids are propeptides, the albumin human and bovine albumin chains contains 585 (25-609) and 583 amino acids (25-607), respectively. The N-terminal tripeptides, DAH or DTH, form the Copper/Nickel binding pocket.

Human Albumin Amino Acid Sequence:
MKWVTFISLLFLFSSAY SRGVFRR DAH KSE VAHRFKDLGE ENFKALVLIA FAQYLQQCPF EDHVKLVNEV TEFAKTCVAD ESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEP ERNECFLQHK DDNPNLPRLV RPEVDVMCTA FHDNEETFLK KYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAA CLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAV ARLSQRFPKA EFAEVSKLVT DLTKVHTECC HGDLLECADD RADLAKYICE NQDSISSKLK ECCEKPLLEK SHCIAEVEND EMPADLPSLA ADFVESKDVC KNYAEAKDVF LGMFLYEYAR RHPDYSVVLL LRLAKTYETT LEKCCAAADP HECYAKVFDE FKPLVEEPQN LIKQNCELFE QLGEYKFQNA LLVRYTKKVP QVSTPTLVEV SRNLGKVGSK CCKHPEAKRM PCAEDYLSVV LNQLCVLHEK TPVSDRVTKC CTESLVNRRP CFSALEVDET YVPKEFNAET FTFHADICTL SEKERQIKKQ TALVELVKHK PKATKEQLKA VMDDFAAFVE KCCKADDKET CFAEEGKKLV AASQAALGL

Bovine Albumin Amino Acid Sequence: (source page)
MKWVTFISLLLLFSSAYS RGVFRR DTH KSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCVAD ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGK YLYEIARRHP YFYAPELLYY ANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLASSARQR LRCASIQKFG ERALKAWSVA RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKE CCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEK LGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLP DTEKQIKKQT ALVELLKHKP KATEEQLKTV MENFVAFVDK CCAADDKEAC FAVEGPKLVV STQTALA

Albumin Complexes: 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 Acid Binding: 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.

Metal Binding: 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.

Mixed Disulfides or Albumin: 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 Binding: 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 and Tryptophan: 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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Albumin Products that Enhance the Growth of Hybridoma, Chinese Hamster Ovary (CHO) and other Mammalian Eucaryotic Cells in Serum-free Cultures:
Product #  Product Name Add to Cart
A1933 Albumin from bovine serum cell culture tested, powder
A4161 Albumin from bovine serum powder, cell culture tested
A4503 Albumin from bovine serum ≥96% (agarose gel electrophoresis), lyophilized powder
A4919 Albumin from bovine serum powder, cell culture tested
A9418 Albumin from bovine serum powder, ≥96% (agarose gel electrophoresis), cell culture tested
A9647 Albumin from bovine serum Heat-shocked fractionate, pH ~7 (1 % (w/v) in aqueous solution), ≥96% (agarose gel electrophoresis), lyophilized powder
A3782 Albumin from human serum Essentially fatty acid free, ~99% (agarose gel electrophoresis), lyophilized powder
A9511 Albumin from human serum 97-99% (agarose gel electrophoresis), lyophilized powder
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Sigma's Cell Culture Media Expert provides in depth discussion of this and other serum-free and protein-free media supplements. The Media Expert contains additional sections on raw materials, component use recommendations, formulation strategies and references. Whenever you have a questions about or problems with your eucaryotic mammalian cell culture media visit the Media Expert for helpful guidance.