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Alteration of its carbohydrate component can therefore potentially affect the biological activity of the glycoprotein, or alter its immunological properties. In practice, however, expression of many normally glycosylated proteins in E. coli generates a protein product whose biological characteristics are indistinguishable from the native product. Furthermore, the glycosylation patterns obtained when human glycoproteins are expressed in non-human eukaryotic expression systems (e.g. animal cell culture) are usually distinct from the glycosylation pattern associated with the native human protein. The glycosylation pattern of human tPA produced in transgenic animals, for example, is different from the pattern obtained when the same gene is expressed in a recombinant mouse cell line. Both these patterns are in turn different from the native human pattern. The tPA from all sources is, however, safe and effective. The clinical significance of altered glycosylation patterns/micro-heterogeneity is best determined by clinical trials. If the product is found to be safe and effective, then routine end-product QC analysis for carbohydrate-based micro-heterogeneity is carried out, more to determine batch-to-batch consistency (which is desirable) than to detect microheterogeneity per se.
Most glycoprotein biopharmaceuticals will exhibit microheterogeneity. Isoelectric focusing of typical batches of the therapeutic monoclonal antibody OKT-3 (Chapter 10) reveals at least four bands, representing four product variations that differ slightly in their carbohydrate content. Likewise, isoelectric focusing of batches of erythropoietin (40 kDa glycoprotein hormone, up to 50% of which is carbohydrate; Chapter 6) typically reveals up to six bands, which again differ only in their carbohydrate content. In any such instance, batch rejection would only be considered if strongly atypical heterogeneity was observed.
Large deviations from normal batch-to-batch glycosylation pattern is most likely caused by the presence of a glycosidase capable of enzymatic degradation of the carbohydrate side-chains. The likelihood of such eventualities may be minimized by carrying out downstream processing at low temperatures, and as quickly as possible.
Stabilizing excipients used in final product formulations
A range of various substances may be added to a purified (polypeptide) biopharmaceutical product in order to stabilize that product (Table 3.22). Such agents can stabilize proteins in a number of different ways. Specific examples include:
• serum albumin. Addition of serum albumin has been shown to stabilize various different polypeptides (Table 3.23). Human serum albumin (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
THE DRUG MANUFACTURING PROCESS 151
Table 3.22. Some major excipient groups that may be added to protein-based biopharmaceuticals in order to stabilize the biological activity of the finished product
Various individual amino acids Various carbohydrates Alcohols and polyols Surfactants
Table 3.23. Various biopharmaceutical preparations for which human serum albumin (HSA) has been described as a potential stabilizer
a- and b-Interferons Tissue plasminogen activator
g-Interferon Tumour necrosis factor
Interleukin-2 Monoclonal antibody preparations
Urokinase g-Globulin preparations
Erythropoietin Hepatitis B surface antigen
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, e.g. for traces of proteases or other agents that could be deleterious to the product. It may also function to directly stabilize the native conformation of many proteins. It has been shown to be an effective cryoprotectant for several biopharmaceuticals (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 is now discouraged, due to the possibility of accidental transmission of blood-borne pathogens. The use of recombinant HSA would overcome such fears.