Removal of dna fragments in mrna production process

Abstract

The present invention describes methods of removing DNA from an RNA transcript during the mRNA production process. The method embodies procedures for obtaining an in vitro transcription product, and removing any DNA from the product. The DNA can be removed by adding either free DNase or a resin containing immobilized DNase to the product, and recovering the RNA transcript. Alternatively, the DNA template used in the in vitro transcription reaction is labeled. After transcription, the product is applied to a resin that is configured to bind the label, and the RNA transcript is recovered. To detect whether any residual impurities are left in the RNA transcript product, the product is subjected to nuclease digestion and subsequently to liquid chromatography-tandem mass spectrometry analysis to quantitate any residual DNA. The present invention demonstrates efficient and effective methods of isolating an RNA transcript from an in vitro transcription product.

1 - 34 . (canceled) 
     
     
         35 . A method for removing a DNA template from a sample comprising the DNA template and an RNA transcript, the method comprising:
 (a) providing the sample comprising the DNA template and the RNA transcript;   (b) digesting the DNA template by contacting the sample with a DNase enzyme, thereby producing a sample comprising a digested DNA template and the RNA transcript; and   (c) separating the digested DNA template from the RNA transcript by a process selected from the group consisting of centrifugation and ultrafiltration, precipitation, and liquid chromatography.   
     
     
         36 . The method of  claim 35 , wherein the liquid chromatography comprises strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and/or hydrophobic interaction HPLC (HIC-HPLC). 
     
     
         37 . The method of  claim 35 , wherein the RNA transcript is the product of in vitro transcription using a non-amplified DNA template. 
     
     
         38 . The method of  claim 35 , wherein the RNA transcript is a full-length RNA transcript. 
     
     
         39 . The method of  claim 35 , wherein the RNA transcript comprises chemically modified mRNA. 
     
     
         40 . The method of  claim 35 , wherein the RNA transcript is from 100 nucleotides to 10,000 nucleotides in length. 
     
     
         41 . The method of  claim 35 , wherein the DNase enzyme is DNase I. 
     
     
         42 . The method of  claim 41 , wherein 2 units of the DNase I are added for each μg of DNA template in the sample. 
     
     
         43 . The method of  claim 41 , wherein the DNase I is incubated with the sample at 37° Celsius for one hour. 
     
     
         44 . The method of  claim 35 , wherein the DNase is attached to a solid support. 
     
     
         45 . The method of  claim 44 , wherein the solid support is selected from the group consisting of a solid phase resin, controlled pore glass, polystyrene, polystyrene divinyl benzene, cellulose, polyacrylamide, silica, polymethacrylate, and dextran based polymers. 
     
     
         46 . The method of  claim 35 , further comprising:
 (d) contacting the sample resulting from (c) with a second nuclease under conditions that promote nucleotide digestion to obtain a digested sample; and   (e) analyzing the digested sample of (d) to detect the presence or absence of residual deoxynucleotides.   
     
     
         47 . The method of  claim 46 , wherein the second nuclease is nuclease P1. 
     
     
         48 . The method of  claim 46 , wherein the digested sample is analyzed in (e) by way of liquid chromatography-tandem mass spectrometry (LC/MS/MS). 
     
     
         49 . The method of  claim 48 , wherein the total abundance of deoxynucleotides present in the sample resulting from (c), as assessed by the LC/MS/MS, is less than 500 ppm, less than 300 ppm, less than 250 ppm, less than 100 ppm, less than 50 ppm, less than 25 ppm, less than 10 ppm, less than 5 ppm, less than 1 ppm, or less than 0.2 ppm.