Application of Enzymes in Drug Manufacturing
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Biocatalysis is a green technology, and life cycle analysis shows that the use of recombinant technologies plays a major part in maximising the sustainability benefit of a biocatalyzed process compared to a traditional chemical process. A significant number of marketed pharmaceuticals contain active pharmaceutical ingredients that are manufactured in part using biocatalysis as a key enabling technology. The utilization of biocatalysis is growing due to significant advances in technologies for enzyme discovery, supply, and improvement, as well as an increased focus on applications for chiral drugs and green chemistry. In fact, a relatively large number of pharmaceuticals already on the market contain intermediates produced by biocatalysis. An explosion in the number and quantities of enzymes available to the synthetic organic chemist has made biocatalysis an increasingly attractive and viable manufacturing option. |
Enzyme Source, Quality, and Specification
Recombinant over-expressed (produced at higher concentrations than in native systems) enzymes are used in pharmaceutical synthesis rather than natural enzymes. This decreases biocatalyst cost, maximises selectivity and efficiency, increases standardization and security of supply when compared to enzymes obtained from natural sources. In the safety evaluation of enzyme preparations for the food industry, the toxicologic potential of the production strain is considered the primary concern, and this concern relates to the potential synthesis of orally active toxins by the production strain. The toxicologic potential of production strains may be managed through the establishment of a safe strain lineage, which involves the use of thoroughly characterized, non-pathogenic and non- toxicologic microorganisms that have a history of safe use as a starting point for the generation of improved strains. In setting a specification of a biocatalyst, an understanding of the purity of the enzyme preparation with regard to other proteins that may be catalytically active may be needed. Small changes in the protein sequence around the N terminus may lead to mixtures of closely related enzymes with different catalytic activities.
Processing Issues
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With regard to processing, apart from the enzyme, other additives can be used in biotransformations. Other materials typically added or introduced to enable biotransformation reactions are generally benign including co-factors like Nicotinamide adenine dinucleotide and corresponding phosphate, (NADH, NADPH), and pyridoxal-phosphate, and other materials such as glucose and buffer salts. Metal ions are required by some enzymes and may be added to biotransformation reactions separately or as part of the biocatalyst. Therefore, these metal cofactors, which can include copper, iron, manganese, magnesium, molybdenum, nickel, selenium, and zinc, may require monitoring to determine their fate. |
With regard to storage and reuse of biocatalysts – enzymes, whole cells and related preparations need to be stored under conditions that are known to retain enzyme activity. If stored cold or frozen, then due care needs to be taken when scaling-up, since warm up and hold times maybe significantly different from those in the lab or pilot plant, and the lengths of freeze-thaw cycles can impact biocatalyst performance.The effect of longer cycle times on enzyme activity needs to be known before a batch of biocatalyst is charged to a reaction.
Residues in API and Strategies for Managing Impurities
Potential impurities associated with small molecule APIs manufactured using enzymes include the enzymes themselves, other host cell proteins, DNA, endotoxins, cell wall debris and antibiotics derived from the fermentation and downstream processing of the biocatalyst. Degradation of these potential impurities may result in the formation of additional impurities such as peptides, amino acids, and polynucleotides. Denaturation of biological molecules typically results in precipitation and therefore provides an opportunity for removal by physical methods. Proteins are denatured by a variety of treatments including organic solvents, strong acids or bases, salts, and heat. DNA can also be precipitated by treatment with organic solvents and removed. Filtration is a useful technique for the removal of biological materials that have been precipitated. Filtration is also useful for the removal of high molecular weight materials that are in solution.
Reference
- Wells A S, Finch G L, Michels P C, et al. Use of Enzymes in the Manufacture of Active Pharmaceutical Ingredients—A Science and Safety-Based Approach To Ensure Patient Safety and Drug Quality [J]. Organic Process Research & Development, 2017, 16(12):1986-1993.
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