Stabilizing excipients used in final product formulations

A range of various substances may be added to a purified therapeutic protein in order to stabilize that product (Table 6.7). Such agents can stabilize proteins in a number of different ways, and some specific examples are outlined below.

Serum albumin addition has been shown to stabilize various different polypeptides (Table 6.8). HSA is often employed in the case of biopharmaceuticals destined for parenteral administration to humans. In many cases, it is used in combination with additional stabilizers, including amino acids (mainly glycine) and carbohydrates. Serum albumin itself is quite a stable molecule, capable of withstanding conditions of low pH or elevated temperature (it is stable for over 10 h at 60 °C). It also displays excellent solubility characteristics. It is postulated that albumin stabilizers exert their stabilizing influences by both direct and indirect means. Certainly, it helps decrease the level of surface adsorption of the active biopharmaceutical to the internal walls of final product containers. It also could act as an alternative target, for example, for traces of proteases or other agents that could be deleterious to the product. It may also function to stabilize the native conformation of many proteins directly. It has been shown to be an effective cryoprotectant for several biophar-maceuticals (e.g. IL-2, tPA and various interferon preparations), helping to minimize potentially detrimental effects of the freeze-drying process on the product.

However, the use of HSA in now discouraged due to the possibility of accidental transmission of blood-borne pathogens. The use of recombinant HSA would overcome such fears.

Various amino acids are also used as stabilizing agents for some biopharmaceutical products (Table 6.9). Glycine is most often employed, and it (as well as other amino acids) has been found to help stabilize various interferon preparations, as well as EPO, factor VIII, urokinase and ar-ginase. Amino acids are generally added to final product at concentrations ranging from 0.5 per cent to 5 per cent. They appear to exert their stabilizing influence by various means, including reducing surface adsorption of product, inhibiting aggregate formation, and directly stabilizing

Table 6.7 Some major excipient groups that may be added to protein-based biopharmaceuticals in order to stabilize the biological activity of the finished product

Serum albumin

Various individual amino acids Various carbohydrates Alcohols and polyols Surfactants

Table 6.8 Various biopharmaceutical preparations for which HSA has been described as a potential stabilizer

IFN-a and -P interferons tPA

Tumor necrosis factor Monoclonal antibody preparations y-Globulin preparations Hepatitis B surface antigen

IFN-y IL-2

Urokinase EPO

Table 6.9 Amino acids, carbohydrates and polyols that have found most application as stabilizers for some biopharmaceutical preparations

Amino acids

Glycine Alanine Lysine Threonine

Carbohydrates

Glucose Sucrose Trehalose Maltose

Polyols

Glycerol Mannitol Sorbitol PEG

the conformation of some proteins, particularly against heat denaturation. The exact molecular mechanisms by which such effects are achieved remain to be elucidated.

Several polyols (i.e. molecules displaying multiple hydroxyl groups) have found application as polypeptide stabilizing agents. Polyols include substances such as glycerol, mannitol, sorbitol and PEG, as well as inositol (Table 6.9 and Figure 6.22). A subset of polyols is the carbohydrates, which are listed separately (and thus somewhat artificially) from polyols in Table 6.9. Various polyols have been found to stabilize proteins in solution directly, and carbohydrates in particular are also often added to biopharmaceutical products prior to freeze-drying in order to provide physical bulk to the freeze-dried cake.

Surfactants are well-known protein denaturants. However, when sufficiently dilute, some surfactants (e.g. polysorbate) exert a stabilizing influence on some protein types. Proteins display a tendency to aggregate at interfaces (air—liquid or liquid—liquid), a process that often promotes their denaturation. Addition of surfactant reduces surface tension of aqueous solutions and often increases the solubility of proteins dissolved therein. This helps reduce the rate of protein ch2oh

CH,OH

OH OH

CH,OH

Mannitol

Inositol

Sorbitol ch2-oh

Glycerol Polyethyleneglycol

Figure 6.22 Structure of some polyols that are sometimes used to stabilize proteins denaturation at interfaces. Polysorbate, for example, is included in some y-globulin preparations, cytokines and in some monoclonal antibody-based products.

Although various polysorbates are used, the experience with an EPO-based product (trade-name Eprex) sounds a potential cautionary note in terms of formulation development, as outlined in Box 4.1.

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