Purification of Oligonucleotides using Prep-LC MS/UV in Combination with Improved IP-RPLC Chromatography

Combining two separation methods utilizing internal and external fraction analysis for purification of native single stranded oligonucleotides (ONs) results in faster collection of target compounds with higher purity as compared to conventional methods.

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Event Overview:

In this study, the automatized Agilent 1290 Infinity II system was used for purification of native single stranded oligonucleotides (ONs). The separations were performed with IP-RPLC using dibutylamine (DBA) and tris(hydroxymethyl) aminomethane (TRIS). The IP-RPLC-system allowed for fraction collection triggered with MSD. Results from the combined Preparative and analysis LC-system included an internal fraction analysis. The purity after Prep-LC/UV separation at ambient and elevated column temperatures were studied by an external fraction analysis, where the high separation capability in analytical runs was included. The combined separation methods with instrumental setup with columns, allowed for the target compound to be collected faster and with higher purity, compared to conventional methods.

Key Learning Objectives:

• Improvement of the purification of ONs by combining improved instrumental setup with modern IP-RPLC chromatography.
• Internal analysis of fractions in Prep-LC purifications with both LC-UV and LC-MS detection.
• A high separation capability chromatography allows for more efficient purification of ONs with respect to lengths of Oligonucleotides and possible modifications.

Featured Speaker:

Olof Stålerg, Ph.D.
Senior Development Engineer, Qiagen DNA Synthesis (QDS)
Västerås, Sweden

Olof is a former project leader and coordinator for technical and pharmaceutical projects at the former Astrazeneca (AZ) R&D in Södertälje, Sweden (closure 2012). During his time at AZ, he developed a wide knowledge base of the different stages of drug development as well as extensive cross-functional and global working experience. He has held a wide range of technical positions in different functions within AZ. These include pre-CD* coordinator working closely with discovery in-silico chemists, a laboratory engineer in analytical development and process R&D functions, a separation science specialist, and a laboratory engineer in operations.

During the autumn of 2012, he had a position at Sirius-Analytical in England which included practical laboratory work and instrument development. In 2015, he joined the University of Karlstad and contributed to publishing scientific articles in a variety of international journals. In his current position as a senior development engineer at Qiagen DNA Synthesis in Västerås, he is involved in separation science, validation, and the development of technical and scientific improvements.

He studied at the Biopharmaceutical Science Center at Leiden, Linköping University, Karlstad University and ultimately earned his doctorate from Uppsala University. He has a family with four grown children, ages 20 – 24 and enjoys spending his free time on outdoor activities cross country skiing and road cycling. In fact, much of his leisure time is spent with his road racing cyclist children where one son competes on the Swedish National Team.

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