Pharmaceutical Technology - October 2021


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14 Pharmaceutical Technology APIs, EXCIPIENTS, AND MANUFACTURING eBOOK 2021 P h a r mTe c h . c o m of the process development is to determine whether there is leaching of the enzyme from the substrate, and if so, how much there is (8). The simplest way to immobilize an enzyme is to adsorb it onto the surface of beads or into the pores within them. The beads are covered by a so- lution of the enzyme, and when they are filtered off, the enzymes are held on the surface by a weak attraction such as van der Waals forces and salt bridges. However, as they are only weakly bound to the surface and within the pores of the beads, there is no guarantee the enzyme will not leach out, which is a regulatory issue. For this method of im- mobilization to be appropriate for industrial and commercial manufacturing, work to demonstrate the consistency and reproducibility of the process with no impact on product quality is essential. An alternative would be to use either cationic or anionic beads, similar to an ion exchange resin. If the protein has a net positive or negative charge, then ionic bonds could be formed between the resin and the appropriate amino acids, holding them in place on the surface. The most secure way to attach the enzymes to the beads would be through some form of covalent bond. If the beads have epoxide groups on the surface, these groups are susceptible to nucleophilic attack from amino acid residues such as lysine or serine, form- ing covalent bonds. Alternatively, the beads could be functionalized with amines and can be treated with glutaraldehyde, which acts as a linker between the beads and the enzyme; and any free aldehyde groups that remain can react with nucleophilic side chains. The enzymes might also be encapsulated in alginic beads or even crystallized out of solution and cross- linked with a molecule such as glutaraldehyde to make solid 'lumps' of enzyme called crosslinked en- zyme aggregates (CLEAs) or crystals (CLECs). From an industrial point of view, however, the most viable option is to attach it covalently to the surface of a bead. Covalent bonding does have some challenges. If nucleophilic amino acid residues are close to, or even in, its active site, and these residues bond to the bead, the activity of the enzyme can be dimin- ished as the diffusion of substrates into the active site would be hindered. The covalent bonding can also be done in a more directed manner, potentially by tagging the enzyme at one end and using this tag to attach it to the surface rather than leaving the binding position to chance. This approach would avoid these diffusion problems. There are some instances when immobilization is less advisable, and principally this would be when binding the enzyme to a resin negates the activity it has, compromising the speed and cost benefits. If the enzyme demonstrates poor activity in the first place, it is unlikely that immobilization would en- hance it, and a better, more active enzyme should be considered instead. Additionally, for cheap, off- the-shelf biocatalysts, there is little point in spend- ing resources to immobilize them, particularly for the pharma sector, where speed is often the primary driver for development. There is no definitive measure for how immobili- zation affects an enzyme's activity, other than that it will probably be lower than that of the free enzyme, perhaps as high as 65% as active or as low as 1%. But as the enzyme is recoverable and reusable, an excess can be used in a reaction, on the understanding that it will not be used just once. Other factors to consider are the ease of handling the enzyme, and whether the filtration process to remove the beads is significantly easier than removing a free enzyme from the reaction mixture. APIs

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