(T1430-10-56) Increasing the Productivity of Oligonucleotide Purification through Column Scaling and Method Optimization

Purpose: Isolation of purified oligonucleotides in an efficient, cost-effective manner can be extremely challenging. These challenges include: 1) Selecting a mobile phase that is affordable, readily available and is easy to remove after fractionation. 2) Scaling the UPLC separation to a larger ID column while minimizing the loss in chromatographic resolution. 3) Optimizing all the chromatographic method parameters to ensure each step of the process is done as efficiently as possible. 4) Optimizing the preparative on-column loading per injection and subsequent fraction collection to maximize the purity and recovery based on the project needs. Reverse-Phase UPLC using an ion-pairing system of HFIP-TEA has become the gold standard for oligonucleotide analysis. This column and mobile system capitalize on the benefits of small columns particles, along with the unique ion-pairing properties using HFIP. The result is a rapid efficient method to accurately assess oligonucleotide’s purity. We explain and optimize the process of scaling an oligonucleotide separation using a UPLC method with a HFIP-TEA ion pairing buffer to preparative scale using a 3 cm ID column. We systematically evaluate the components of each step of the process to understand the impact of its variation and its impact on the overall process. The different items tested and varied include mobile buffers, mass and volume column loading and fraction collection.

Methods: The methods used are analytical UPLC and preparative HPLC methods optimized for the separation and isolations of oligonucloetides.  The employ C18 stationary phases of 2.5 um and lower with reverse phase mobile phases of both HFIP:TEA and TEAA.  Optimization of various parameters will be shown.

Results: The initial UPLC HFIP:TEA method on 1.7um C18 particles was converted to an HPLC TEAA method on 2.5um C18 particles.  Only minor loss of the impurities, mainly the N-1 impurity, was observed.  This analytical HPLC method was then scaled to a 30mm ID preparative scales and fractions collected.  Using the original UPLC method to QC the collected fractions, the isolated oligonucleotide was over 95% pure.  To improve the overall productivity multiple method parameters were evaluated and optimized. This include mass load on column, flow rates through out the run and collection timing.  The results demonstrate a 100X increase in the overall productivity compared to the original scaled preparative method.

Conclusion: Isolation of purified oligonucleotides is possible when the appropriate mobile phase and column conditions are selected.  The use of HFIP containing not required for the preparative method.  We demonstrate a RP-HPLC method on a 30mm ID preparative column using TEAA is capable of efficiently generate over 95% pure oligonucleotide, even when the purity is assessed using a HFIP-TEA UPLC method. Optimizing loading, fraction collection and method parameters can improve productivity.  However, the overall productivity is driven by the purity requirement for the project. The final method development produces over 95% pure material at a rate of over 100 mg / hour.  Furthermore increases could be realized by increasing the preparative column and through offline column cleaning and regeneration.

References: Gilar, M. et al., HPLC and UPLC column for Analysis of Oligonucleotides, Waters Application Note.
McCarthy, S. et al., HPLC and UPLC column for Analysis of Oligonucleotides, Waters Application Note
Lefebvre, P. et al., Evaluating the Tools for Improving Purification Productivity, Waters Application Note.

Table 1: Analytical methods using with HFIP:TEA and TEAA



HPLC analysis with TEAA



Figure 1: UPLC analysis with HPIF:TEA.