LAMP and RT-PCR accelerated stability tests
Hi all,
I am successfully producing RT-qPCR and LAMP enzymes in Brazil. The following steps consist of registering these products as IVD in our “FDA,” but we need to perform first the stability tests.
My question is:
Does anyone know any Norm or official method to do stability tests in molecular biology proteins? I have seen some protocols using 50C-60C temperatures to perform the accelerated tests. I am afraid that this general protocol would not be suitable for these proteins. If those enzymes and their mixes do not stand for longer than a week, these kits will have a very short expiry date.
Let me know if you have a hind to do that.
Cheers,
Robson – ECRA Biotec
1 Like
Hey Robson
Typically accerated studies are also accompanied by real time data as well for a LDT or FDA submission. The temp does not have to be 50-60 this is only used for the most robust of enzymes. Accelerated studies can be done with 4C or any temp you like 25C or 30 or 37C. Depends on the enzyme, reagents, cofactors, pH, ions, and redox potential for the reagents in question. Typically we would run some of these. For some enzymes they just can do a high temp (like Reverse Transcriptase) and you HAVE to do the real time study. You can also submit a vendors data for the enzyme or system with the note that you are running both studies and will present the data when finished. SO the important tidbit you are looking for the Arrhenius Equation is the conversion constant you are looking for to help you plan your study. More info here --> https://www.rmtesting.com/accelerated-aging-calculator-medical-device-packaging-testing.php#:~:text=The%20common%20and%20conservative%20means,claims%20and%20document%20expiration%20dates. Plan you intervals to double out to a year (T0, T = 1wk, T = 2wk, T= 4wk, T= 8 weeks, T= 4 Months … fill in some other points like 6 months out to a year and beyond to see it really crash.) and over plan the number and amount you are going to need for the test. IT SUCKS to mess up a real time or accelerated study because you run out of reagents in the fridge!! Trust me OVER do it with extra data point replicates and extra reagents just double or triple what you think you need don’t even cut it close over do it. I do not have the energy or ability to share a plan with you (NDA’s and junk). I wish you a lot of luck in this. ALSO if you are leveraging a vendor like NEB talk to them about how they did their study they will share their knowledge and possibility the design. OH, one cool thing we found for an enzyme was by adding a little extra or boosting the activity we sustain the activity drop by quite a bit and still hit our target out to a year and beyond so if you are not making the cut off for activity you you can have a 1.2x concentration or 1.5x concentration that will maintain your desired active threshold to a year or what ever your target is. You might be wasting enzyme but it pays off in the end with a longer shelf life. Good Luck. Sorry for the typos Eric