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9/20/2022 8:15:23 PM 

About WuXi STA's One-Stop Biocatalysis Platform

Mid-sized US pharma company asked for a more economical method for synthesis of an API intended for a high volume therapeutic as their novel drug showed good activity as it progressed through clinical trials.

The chiral amine shown in Fig 1. was originally made by a 5-step chemical synthetic route. The overall isolation yield was low, at ~30% and the cost of the amine raw material accounted for ~80% of the total cost of the API. The client told us there was an urgent need to lower the cost of the amine, as its high cost could be prohibitive to a successful commercial launch of the API. To protect our client's IP protection, we can only give a general structure here.

WuXi STA Solution:
Clinical stage solution
Risk analysis indicated it was very probable that a 1-step enzymatic process could make the desired chiral amine directly from the corresponding ketone for 5 steps. This would be a much more cost-effective process if successful.
Our team rapidly screened *150 commercial enzymes in our library for this chemical transformation. We found one enzyme that could give a 28.5% conversion with excellent chiral purity of 99% enantiomeric excess (ee). However, the enzyme loading was 1:1, which is high considering the high cost of the commercial enzyme.

We decided to optimize the reaction conditions to see if we could increase the conversion and lower the enzyme loading to a more acceptable level.

First, we quickly screened amine donors and found isopropylamine gave the best result. However, isolation of the product was difficult due to excess isopropylamine in the system. To facilitate the product isolation, we decided to explore the use of alanine as the amine donor, even though the initial screening result was not the best.

We found that we could use a multi-enzymatic cascade adding AADH/GDH/NH4, to regenerate alanine in-situ (to avoid inhibition of high level of newly generated pyruvic acid). This 3-enzyme process pushed the conversion to ~78%.

We also found if the substrate is added dropwise, a * 90% conversion could be obtained.

This optimized condition was scaled up to 40 g (Fig 2). The ee of the product was still excellent (* 99%) and isolation yield was acceptable at ~65%. This demonstrated that the enzymatic route could lower the cost of the amine by ~30%, even though the cost of enzymes was still high.

Commercial stage solution
As the project moved to the later stage and batch sizes increased, our client still desired to further lower the cost of the API. To make the process more efficient we performed more iterations of screening. We found a wild-type enzyme with some activity and then proceeded to perform enzyme evolution. We were delighted to find that we could improve the enzyme activity by ten times, and the ee reached * 99% within two rounds of mutations (Fig 3). The enzymatic process with the new low-cost enzyme was successfully scaled up to *140 kg, with a 75% lower product cost.

Biocatalysis is becoming more important for the manufacture of complex synthetic molecules thanks to the power of enzyme evolution to replace costly, lengthy, and unsafe processes and eliminate or reduce environmentally harmful waste products.
This case study showed that our expert process development scientists, equipped with a large enzyme library, high throughput screening capability, and ample fermentation capacity, could successfully improve the efficiency and lower the product cost of an API that may have never been commercially feasible, leaving patients with unmet needs.
About WuXi STA's One-Stop Biocatalysis Platform

About WuXi AppTec One-Stop Biocatalysis Platform Link:www.stapharma.com

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About WuXi STA’s One-Stop Biocatalysis Platform