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biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
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146 BIOPHARMACEUTICALS
Figure 3.22. Oxidation of (a) methionine and (b) cysteine side-chains, as can occur upon exposure to air or more potent oxidizing agents (e.g. peroxide, superoxide, hydroxyl radicals or hypochlorite). Refer to text for specific details
THE DRUG MANUFACTURING PROCESS 147
I I
S-------------------S
Figure 3.23. Diagram representing the molecular process of intra-chain (a) and inter-chain (b) disulphide exchange. Refer to text for specific details. (-O- = amino acid residues in polypeptide)
Oxidation of methionine is particularly favoured under conditions of low pH, and in the presence of various metal ions. Methionine residues on the surface of a protein are obviously particularly susceptible to oxidation. Those buried internally in the protein are less accessible to oxidants. Human growth hormone (hGH) contains three methionine residues (at positions 14, 125 and 170). Studies have found that oxidation of methionine 14 and 125 (the more readily accessible ones) does not greatly effect hGH activity; however, oxidation of all three methionine residues results in almost total inactivation of the molecule.
Oxidation can be best minimized by replacing the air in the head space of the final product container with an inert gas such as nitrogen, and/or the addition of antioxidants to the final product.
Disulphide exchange can also sometimes occur and prompt a reduction in biological activity (Figure 3.23). Intermolecular disulphide exchange can result in aggregation of individual polypeptide molecules.
Alteration of glycoprotein glycosylation patterns
Many proteins of therapeutic value are glycoproteins, i.e. display one or more oligosaccharide chains covalently attached to the polypeptide backbone. Examples of such proteins include immunoglobulins, blood clotting factors, ^-antitrypsin and some interferons. Analysis of the carbohydrate composition of glycoproteins reveals the presence of a variety sugars, including D-galactose, D-mannose and L-fucose (neutral sugars), the amino-sugars; N-acetylglucosamine
148 BIOPHARMACEUTICALS
Figure 3.24. Monosaccharides that commonly constitute the carbohydrate portion of glycoproteins. Refer to text for details. Note that individual hydrogen atoms attached to the core ring structure are omitted for clarity of presentation
and N-acetylgalactosamine, and acidic sugars (e.g. sialic acid) (Figure 3.24). These sugar chains are attached to the protein backbone via two common covalent bond types (Figure 3.25):
1. O-glycosidic linkages, in which the sugar side chain is attached via the hydroxyl group of serine or threonine, or sometimes modified amino acids such as hydroxylysine.
2. N-glycosidic linkages, in which attachment is via the amino group of asparagine (rarely, attachment via an S-glycosidic linkage involving cysteine residues can also occur).
Carbohydrate side chains are synthesized by a family of enzymes known as glycosyltrans-ferases. For any glycoprotein, the exact composition and structure of the carbohydrate side-
THE DRUG MANUFACTURING PROCESS 149
Figure 3.25. N-linked (a) versus O-linked (b) glycosylation. Reproduced by permission of John Wiley & Sons Inc. from Walsh (2002)
chain can vary slightly from one molecule of that glycoprotein to the next. This results in microheterogeneity, which can be directly visualized, e.g. by isoelectric focusing (discussed later). Virtually all therapeutic glycoproteins, even when produced naturally in the body, exhibit such heterogeneity; e.g. two species of human interferon-g, of molecular mass 20 and 25kDa, respectively, differ from each other only in the degree and sites of (N-linked) glycosylation. The sugar chains on glycoproteins can serve a number of different functions, including:
• in some cases the carbohydrate component plays a direct role in mediating the glycoprotein’s biological activity, e.g. removal (or extensive alteration) of the carbohydrate component of human chorionic gonadotrophin (hCG; Chapter 8) significantly decreases its ability to induce characteristic responses in sensitive cells. Desialylated erythropoietin (EPO; Chapter 6) also exhibits very little biological activity in vivo;
• the carbohydrate component may play a recognition role in some cases, e.g. sugar residues of some glycoproteins can play an essential role in processes such as lysosomal targeting and cell adhesion events;
150 BIOPHARMACEUTICALS
• the carbohydrate component serves to substantially increase the solubility of several glycoproteins;
• the carbohydrate component helps regulate the biological half-life of some serum glycoproteins. It increases the serum half-life of some glycoproteins by decreasing the rate of clearance from the serum. Furthermore, modification/partial degradation (i.e. asialogly-coproteins) of the carbohydrate component can accelerate removal of the protein from the serum;
• the carbohydrate component probably exerts a direct stabilizing influence upon the conformation of many glycoproteins.
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