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#### jeffmoonchop

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##### Reduce Ethanol Concentration
« on: October 24, 2018, 01:14:47 PM »

Hi all, I'm working on developing a nanoparticle formulation where two components, one in water (mRNA) and one in ethanol (lipid) is mixed. I need to run it through a TFU but before I do I need to reduce the volume of ethanol to below 17%. Does anyone have any ideas of how to do this without breaking my nanoparticles? i.e. gently, without boiling, etc.

At the moment I've been trying dilutions, blending then doing two separate dilutions. This reduces the ethanol concentration enough but Im left with large volumes and low particle concentration.

My background is in chemistry so not used to being so careful with biological material.

cheers
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#### P

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##### Re: Reduce Ethanol Concentration
« Reply #1 on: October 24, 2018, 10:35:32 PM »

Roto vap or reduced pressure evaporation? The reduced pressures will allow you to avoid the higher temps you are trying to avoid.

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Tonight I’m going to party like it’s on sale for $19.99! - Apu Nahasapeemapetilon #### wildfyr • Global Moderator • Sr. Member • Mole Snacks: +130/-9 • Offline • Posts: 1150 ##### Re: Reduce Ethanol Concentration « Reply #2 on: October 25, 2018, 04:52:13 AM » If you don't even want to rotovap at room temp, you could just let it sit out on the bench in a beaker for a while. Ethanol is pretty volatile. Logged #### Babcock_Hall • Chemist • Sr. Member • Mole Snacks: +234/-16 • Offline • Posts: 3622 ##### Re: Reduce Ethanol Concentration « Reply #3 on: October 25, 2018, 05:46:17 AM » Or perhaps one could use a gentle stream of nitrogen gas. Logged #### jeffmoonchop • Full Member • Mole Snacks: +9/-1 • Offline • Gender: • Posts: 102 ##### Re: Reduce Ethanol Concentration « Reply #4 on: October 25, 2018, 07:00:59 AM » Thanks for all your suggestions I think they're great. However, my volumes may increase up to 30L is there a way to scale up reduced pressure evap? Letting it stand for evap probably wont work with the volumes I may use. It would need to be kept sterile too due to unstable mRNA. Logged #### Yggdrasil • Retired Staff • Sr. Member • Mole Snacks: +482/-21 • Offline • Gender: • Posts: 3199 • Physical Biochemist ##### Re: Reduce Ethanol Concentration « Reply #5 on: October 26, 2018, 05:14:21 AM » Would a desalting column or dialysis work for buffer exchange? Alternatively, one could perform alternating rounds of dilution/concentration in centrifugal concentration units. Logged #### jeffmoonchop • Full Member • Mole Snacks: +9/-1 • Offline • Gender: • Posts: 102 ##### Re: Reduce Ethanol Concentration « Reply #6 on: October 26, 2018, 05:52:29 AM » Would a desalting column or dialysis work for buffer exchange? Alternatively, one could perform alternating rounds of dilution/concentration in centrifugal concentration units. I currently use either TFU for larger volumes or dialysis for smaller volumes but the concentration of ethanol needs to be low to do that due to the affect on the nanoparticles. So currently Im just diluting it but I'm trying to eliminate the dilution step by getting rid of the ethanol another way. Logged #### Yggdrasil • Retired Staff • Sr. Member • Mole Snacks: +482/-21 • Offline • Gender: • Posts: 3199 • Physical Biochemist ##### Re: Reduce Ethanol Concentration « Reply #7 on: October 26, 2018, 06:21:20 AM » I have no idea what TFU means. Proteins and biological nanoparticles (viruses) can be precipitated from solution by PEG precipitiation (http://cshprotocols.cshlp.org/content/2006/1/pdb.prot4311). Could you use that to precipitate your nanoparticles, so that you can resuspend them in a buffer without ethanol? Logged #### jeffmoonchop • Full Member • Mole Snacks: +9/-1 • Offline • Gender: • Posts: 102 ##### Re: Reduce Ethanol Concentration « Reply #8 on: October 26, 2018, 08:03:55 AM » Sorry its Tangential flow ultrafiltration. Basically swapping the buffer out, but only after the ethanol concentration is low enough. Precipitation is interesting, I'm not sure how gentle that would be for the particles though. I'll look into it thanks. Logged #### P • Full Member • Mole Snacks: +63/-15 • Offline • Gender: • Posts: 629 • I am what I am ##### Re: Reduce Ethanol Concentration « Reply #9 on: October 29, 2018, 11:40:57 PM » my volumes may increase up to 30L is there a way to scale up reduced pressure evap? Do it in batches.. or just leave the lid off the 30L container for a few days - as someone above said - it's pretty volatile and will evaporate easily. If you leave in a bath rather than a bucket then it will evaporate faster due to the surface area of the batch or tray compared to the bucket/beaker. Logged Tonight I’m going to party like it’s on sale for$19.99!

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#### jeffmoonchop

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##### Re: Reduce Ethanol Concentration
« Reply #10 on: October 30, 2018, 01:09:35 PM »

Time is also a factor. I cant just let it sit for a few days. The mRNA would degrade. Would prefer to rotevap it all in one batch but it seems to be the best idea so far. Cheers
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#### P

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##### Re: Reduce Ethanol Concentration
« Reply #11 on: October 31, 2018, 01:58:26 AM »

A 30 L round bottomed flask in a heating mantle with a vacuum distillation rig then?  You can heat very gently as the reduced pressure will be lowering the boiling point greatly. Won't take that long if you pull a decent vacuum.
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