(281f) Liquid Phase Oligonucleotide Synthesis with Membrane Separation for Efficient Large Scale Manufacturing

Liquid Phase Oligonucleotide Synthesis with Membrane
Separation for Efficient Large Scale Manufacturing

Danilo
Cuccato^a, Jack H. J. Cordrey^a, Piers R. J. Gaffney^a,
Patrizia Marchetti^a, Jeong F. Kim^a, Daniela Negru^b,
Mike S. Anson^b, Andrew G. Livingston^a.

^aDepartment of Chemical
Engineering, Imperial College, London, SW7 2AZ, UK

^bGSK, Medicines Research
Centre, Gunnels Wood Road, Stevenage, Herts, SG1 2NY, UK

Abstract

Oligonucleotides
(oligos, 18 to 25-mers) are difficult to prepare at high purity using solid
phase oligo synthesis (SPOS) on scales greater than 1 kg, and extensive
chromatography of the crude full-length oligo is required to reach acceptable
purity. We have developed a liquid phase oligo synthesis (LPOS) strategy that allows
in-line monitoring of coupling efficiency throughout the synthesis, so that any
incomplete reactions so detected can be driven to completion. After each chain
extension the growing oligo is separated from reaction debris using a two-stage
nanofiltration apparatus. To enhance the separation, three oligos are linked to
a central hub in a three arm oligo-star (3ⁿ). The
purified oligo-star is then concentrated, again using molecular sieving, ready
for the next chain extension. This process enables LPOS in standard chemical
process plant, and for building block usage to be minimised. A new class of
membranes was developed that efficiently retains oligo-stars but permeates
large reactant debris (MW ca. 600 Da) in strong organic solvents over
more than 19 synthesis cycles. The synthesis cycle was repeated up to a 20-mer (4)
using a 5’-methoxyisopropyl protecting group (2, P = Mip).
Current work, now using commercial phosphoramidites (2, P
= Dmtr), seeks to reach greater than 90% crude purity. This technology is being
commercialised by EXACTMER, a new start-up from Imperial College London.