Oligos 201 – Purification and Isolation

Choosing the Best Purification Method: Method Scouting Wizard for Oligo Separation

Synthetic ONs are a class of compounds that have gained increasing interest over the last few years because of their use in biochemical research and as pharmaceuticals. (1) The process of synthesizing ONs has become much more efficient since its implementation in the 1980s. However, even with 99% coupling efficiency, a 25mer ON synthesis will yield less than 80% of the desired product. By-products are, for example, truncations of the target ON that have a sequence length of N–1, N–2, etc. Separating these impurities from the target molecule becomes more challenging with increasing sequence length.(2) Analytical HPLC method development is important in the chemical and pharmaceutical industries. Typically, a set of columns is tested in combination with a set of mobile phases. The Agilent 1290 Infinity II Method Development Solution offers a highly flexible system for one through eight columns as well as, solvent selection valves can be clustered with the pump to enhance the selection of available mobile phases

Presenter: Lori Sandford (HPLC Applications Scientist, Agilent Technologies, Inc.)

Lori Sandford is an HPLC Applications Scientist with Agilent Technologies, Inc. in Wood Dale, IL. Lori has been working at Agilent Technologies supporting HPLC instrumentation since 2010, including 2 years with Varian working as an HPLC Specialist.

Prior to Agilent, she was the Global Technology Manager for Prep and Process Chromatography at W. R. Grace/Alltech. Her career started at G.D. Searle/Pfizer in 1998 as a chromatography specialist in the Prep and Process Chromatography group supporting both Medicinal and Process Separations. Lori attended Florida State University where she received her degree in Chemistry. When not tinkering in the lab, Lori enjoys time at home with her 2 teenage boys shuttling from hockey rink to Lacrosse field, enjoying concerts or planning her next scuba adventure.

Scaling for Oligo Purification: Loading Study and Scale Up Calcs from 4.6 x 150mm to 21 x 150mm Column

Once separation conditions have been identified, additional studies are carried out on analytical scale to examine the effects of concentration and/or injection volume on column loading, aiming for the best balance of purity, yield and throughput. With the help of a method scale up calculator, the optimized conditions can be transferred to columns of larger dimensions. With the addition of fraction collection, isolation of pure material is enabled. All experiments discussed here are conducted using Agilent PLRP-S columns, which are available in both analytical and preparative dimensions, thus enabling easy scale-up and high reproducibility from analytical to preparative purification conditions.(2)

Presenter: Regina Black (LC Applications Scientist, Agilent Technologies Inc.)

Gina joined Agilent over 2 years ago and brought with her a wealth of experience in the pharmaceutical industry. She began her career at Merck as a synthetic chemist and became a subject matter expert in preparative HPLC, eventually joining the Separations COE, supporting both discovery and process development efforts. After twenty-two years at Merck, Gina moved to Bristol-Myers Squibb where she served as a site lead for their Single Compound Purification group, using a highly automated workflow to analyze, purify and deliver final products for biological testing. Gina then moved back into large scale separations and enabling technologies supporting Chemical Development at BMS. Over the years, Gina has developed an extensive background in both reverse phase and normal phase HPLC, SFC and IEX methodology.

Oligonucleotide Purification Using Mass Based Fraction Collection

Using mass-based fraction collection can easily facilitate the collection of single-stranded RNA ON from complex mixtures. The 1290 Infinity II Preparative LC System features a binary pump that is capable of precise gradient mixing even at high flow rates, which improves reproducibility and confidence in the results. Titanium pump heads are compatible with a wide pH range and salt concentration, stretching the range of applications that can be run on the instrument. Choosing the correct trigger ion of the desired compound will lead to better fraction collection resulting in purer final products. The combination of the 1290 Infinity II Preparative LC System with the LC MSD XT single quad mass spectrometer provides an ideal purification capability.

Presenter: Patrick Cronan (LC Applications Scientist, Agilent Technologies, Inc.)

Patrick Cronan is an LC applications Scientist with Agilent Technologies. He is an accomplished analytical chemist with over 20 years of experience in the pharmaceutical and Biopharma industries; has facilitated method transfer from HPLC to UHPLC while using LCMS to characterize large and small molecule drug-like compounds. Patrick has also worked extensively with Agilent’s 2D Chromatography system developing methods to increase throughput of biologic separations by coupling multiple techniques like SEC x IEX.

(1) Roberts, T. C.; Langer, R.; Wood, M. J. A. Advances in Oligonucleotide Drug Delivery. Nat. Rev. Drug Discov. 2020, 19, 673–694

(2) Rieck F., Agilent Technologies, Purification of Single-Stranded RNA Oligonucleotides Using High-Performance Liquid Chromatography, Application Note 2021.