Speciality Chemicals Magazine MAY / JUN 2024 | Page 37

GREEN CHEMISTRY

Ligase technology

Recently , RNA-based therapeutics and vaccines have emerged as a powerful new treatment and prevention options for severe disease and illness limiting the human life span . RNA ligases are a powerful enzyme class , enabling the industrialisation and scalethe statistic does not show is the diversity of the publications , but it demonstrates that biocatalysis is no longer a niche or emergent technology .

Nowadays , most pharmaceutical CDMOs have dedicated biocatalysis groups . Those like Almac who have been leading this trend for over 15 years have amassed huge libraries of enzymes from many classes to facilitate the need for speed in early clinical development .

Almac has also invested in metagenomics , a technique to access more of nature ’ s enzyme diversity , helping to advance enzyme engineering from a place not well suited to quick problem solving to a better place , where the power of computation and rational prediction replaces the need for slow and costly handling of tens of thousands of colonies and clones . 6

Regularity & novelty

While many enzyme classes have been investigated and many more will follow , Almac statistics indicate their application frequency and magnitude . The top spot is still held by CRED enzymes , with the wider reductase category also featuring in manufacturing with amino acid

Figure 2 - Almac ’ s biocatalysis multikg to tonne scale-up count dehydrogenases ( AADHs ) and imine reductases .

Interestingly , the fragility of oxidative enzymes has been overcome for Bayer-Villiger-monooxygenases ( BVMO ), and scale-up applications now range from small- and largescale preparation of non-GMP building blocks and regulatory starting materials to late-phase and commercial API manufacture with biocatalytic route-defining steps .

Reductases are the most frequently used enzyme class because of their reliability and predictability , making their selection a low-risk option . AADH enzymes demonstrate the versatility and substrate promiscuity of this class by enabling access to unnatural amino acids , which have become ever more frequent building blocks for new drugs , and feature as key ingredients in the trend towards larger drug molecules , such as peptides .

Number of completed processes at manufacturing scale

Enzyme class / type non-GMP GMP Carbonyl reductase ( CRED / KRED / ADH 22 9 Hydrolase ( lipase / esterase / protease 10 2 Amino acid dehydrogenase ( AADH ) 6 2 Imine reductase ( IRED ) 3 3 Transaminase ( TAm ) 5 1 Baeyer villager monooxygenase ( BVMO ) 1 2 Lipoxygenase ( LPO ) 2 2 Ligase / kinase ( BOOST ) 7 - P450 / other hydroxylate / tyrosinase / DO 5 2 Cyclase 2 - up of these highly diverse large molecular entities . 7

Whilst established solid-phase supported chemical synthesis methods can access a huge structural variety and molecular size of oligonucleotides , the cost of making long RNA this way is prohibitive for mass market application , due to the problematic nature of highly similar impurities and the need for preparative chromatography for clean-up .

RNA ligases can recognise their substrates among similar compounds and provide ligation and purification in one reaction step . Their rapid engineering with today ’ s smart mutant library toolbox makes catalyst tuning towards the acceptance of unnatural , such as methylated or fluorinated , nucleosides a routine undertaking .

Outlook

From a biocatalysis expert CDMO perspective , the trends of today that will become the treatment agents of tomorrow include :

• Further roll-out of robust chiral technologies , including more and more oxidation , for instance producing chiral sulfoxide pharmacophores

• Overcoming equilibrium problems in bio-amination technology by developing high affinity co-enzyme systems

• Further development of peptide ligation to enable mass production of peptide APIs with unnatural fragments that cannot be produced by recombinant fermentation . Innovations in co-factor regeneration are also emerging to help to reduce cost . 8 , 9 ●

* - The author would like to thank OPR & D editor Kai Rossen and his ACS Paragon team for supplying the biocatalysis publication statistics

Stefan Mix

ASSOCIATE DIRECTOR CHEMISTRY – BIOCATALYSIS

MAY / JUN 2024 SPECCHEMONLINE . COM

37