HIGH POTENCY APIS
Toxin-linker
( high potent )/ -- -+ TBTU / solution
Temperature control unit
Figure 2 - Flow chemistry process of PDC , TBTU & toxinlinker coupling reaction
Other starting -- ---ti materials
Temperature control unit
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Reaction 1 @ 20 ~ 25 ° C
Peptide / __ ___,. solution
Temperature control unit
Reaction 2 @ 5-10 ° C
Crude PDC decomposition of the activated ester intermediate of the toxin-linker-TBTU complex . While TBTU is a common assembly reagent in peptide ( and oligonucleotide ) conjugation , it does have limitations , primarily the fact that the ester bond between TBTU and the linker is unstable , subject to nucleophilic attack . Thus , even when we carefully design a process , with all of the reagents and solvents selected to be non-nucleophilic , one factor still remains : the toxinlinker itself , which is likely to cause side reactions .
In this reaction in particular , the toxin-linker also had three nucleophilic hydroxyl groups ( -OH ), compounding the problems . This nucleophilic property resulted in intramolecular condensation when these alcohols attacked the TBTU ester ( Figure 1 ) and this side reaction significantly dropped the yield and generated impurities .
This was not a problem in the gramscale synthesis when the reaction time was short and the concentration of the toxin-linker-TBTU complex remained low . During the scaledup manufacturing at kg-scale , however , with increased reaction time and local concentration , the side reaction between the complex itself significantly increased , posing impurity and yield challenges .
Because of the special structure of the toxin , TBTU degradation was difficult to resolve in large-scale batch manufacturing . To overcome this challenge , the team at WuXi STA applied flow chemistry to avoid degradation conditions .
The starting materials were fed into one-way pipe reactors at a precisely controlled time and in a precisely controlled quantity . The reactions were fully automated and took place in an enclosed system , minimising human exposure to highly potent compounds .
After adjusting to the ideal reaction temperature , the toxin-linker-TBTU complex was generated continuously in the reaction stream ( Figure 2 , reaction 1 ). The peptide was then fed into the system immediately and coupled with the toxin-linker-TBTU complex ( Reaction 2 ).
The concentration of the complex was always limited to a low level by conjugating with the peptide in the flow mode before any side reaction could take place . Moreover , compared with the batch mode , the reaction temperature could be increased to 10 ° C , accelerating the reaction speed twofold .
As a result , seven grams of crude PDC were manufactured every hour with a high yield of over 76 %. With follow-up isolation and purification , multiple kilograms of the PDC API were delivered with more than 97 % purity .
Outlook
Flow chemistry technology was successfully employed in this project to address the scale-up challenge of this PDC drug . With the fully enclosed reactors and automated system , it minimised human exposure to highly potent compound .
The flow mode reduced the local concentration of the toxin-linker- TBTU complex . It also shortened the reaction time with increased temperature , so the condition for side reactions was avoided , showing unique advantages compared with the batch mode .
As PDC drugs involving powerful cytotoxins continue to thrive , it is foreseeable that flow chemistry will play an increasingly important role in enabling these challenging therapeutic modalities . ●
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Dr Youchu Wang
SENIOR VICE PRESIDENT , HEAD OF API EARLY PHASE DEVELOPMENT
WUXI STA info @ wuxiapptec . com www . stapharma . com www . sentry-equip . com
JUL / AUG 2023 SPECCHEMONLINE . COM
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