For decades, the complexity of albumin has been researched extensively, yet many manufacturers and users of the protein have treated it more as a commodity. Because albumin has been readily available, suppliers and purchasers alike have frequently relied on more obvious measures of “purity” and other minimal release criteria to make their decisions. If a lot does not perform well in practice, the typical supplier's response has been to investigate the manufacturing process for deviations, then correct them if found and make another lot. This is a costly strategy for both manufacturer and purchaser — especially if the “corrected” lot still doesn't work. So what's the answer? We can begin with an appreciation of albumin's heterogeneity, then determine the effects of purification processes on its properties, and correlate those properties to the product application.

Albumin is the most abundant serum protein. It serves several functions in vivo: e.g., binding and transport of fatty acids, hormones, and metal ions; maintenance of osmotic pressure and pH; and binding of exogenous toxins and products of lipid oxidation (1). Over time, development of large-scale purification methods have translated those functions into diagnostic, cell culture, and microbiological applications. It is important to note, however, that purification procedures can promote molecular changes and thereby add to the already complex nature of albumin. That increase in heterogeneity can cause unpredictable performance in vitro. Consequently, it is important for suppliers to understand how the process modifies the end product — and for buyers to consider the potential effects that process-induced modifications can have on the performance of their process. Relying strictly on published product specifications is often insufficient when making a decision to purchase.

PRODUCT FOCUS: ALL BIOLOGICS
PROCESS FOCUS: PRODUCTION
WHO SHOULD READ: MANUFACTURING, PROCESS DEVELOPMENT, QA/QC
KEYWORDS: CONTAMINATION, MEDIA, ANALYTICAL METHODS, TSE, RAW MATERIALS
LEVEL: INTERMEDIATE

THE ALBUMIN MOLECULE

Bovine and human albumins are carbohydrate-free monomeric proteins composed of three homologous domains (2,3). Estimated molecular weights are 66,267 and 66,439 Daltons, respectively (3). Their amino acid sequences are ~80% homologous, with bovine albumin composed of 582 amino acid residues and human albumin 585. Each contains 16.3–16.5% nitrogen (3). There are 35 cysteine residues involved in 17 intramolecular disulfide bridges, which contribute to overall conformation and stability (3).

A Cys34 residue is positioned at the solvent interface of each albumin molecule and has a free sulfhydryl (SH) group that is prone to oxidation and mixed disulfide formation. Depending on the age of an albumin preparation (and the method by which it was purified), slight differences have occurred in the SH/albumin molar ratios and extinction coefficients. For example, Janatova found a correlation between the SH/albumin ratio and the extinction coefficient: 6.67 for a freshly prepared albumin and 6.82 for an aged mercaptalbumin preparation with an SH/albumin molar ratio of 0.65 and 0.72, respectively (4). In vivo, the free sulfhydryl group is susceptible to oxidation by cysteine or glutathione, forming mixed disulfide bonds (2).

Fatty acids represent 80% of all the lipids bound to albumin. These bound fatty acids affect its conformation, electrophoretic mobility, and isoelectric point (pI). As a defatted albumin molecule is resaturated with fatty acids, its volume increases, dipole moment decreases, electrophoretic mobility increases, and pI decreases. It is estimated that 1–2 moles of long-chain fatty acids are bound to circulating albumin with a maximum fatty acid/albumin molar ratio of six — the same value that can be achieved in vitro (3). The first and second binding sites have the highest affinity and the greatest influence on the protein structure. However, Chen found the average fatty acid content of several commercially available bovine albumins to be ~0.5 mol/mol (5).

The following fatty acids are normally bound to albumin in vivo and are listed here in descending order of their affinity for albumin: oleate, stearate, linoleate, palmitate, and arachidonate. In addition to fatty acids, other ligands have varying affinity for albumin. Among its significant ligands are calcium, copper, zinc, prostaglandin, cortisol, testosterone, bilirubin, hematin, and thyroxine (3). Given that many of those ligands remain bound to purified albumin, it may be advisable for purchasers to consider measuring the relevant ones to ensure their protein's optimal performance in specific applications. For example, Spector found that even trace amounts of fatty acids bound to human albumin will affect its ability to interact with drugs, dyes, detergents, and other potential binding ligands (6).

Another feature of albumin is its propensity to polymerize. It is common to find commercially available lyophilized albumins with high–molecular-weight polymers. That polymerization is traceable to specific steps in the manufacturing process that cause protein unfolding: acidic pH conditions (7,8), exposure to a source of energy (e.g., high temperatures (8,9), radiation (9,10), and dehydration caused by an organic solvent (11).