Synthesis of "Nereid," a new phenol-free detergent to replace Triton X-100 in virus inactivation.

In the 1980s, virus inactivation steps were implemented into the manufacturing of biopharmaceuticals in response to earlier unforeseen virus transmissions. The most effective inactivation process for lipid-enveloped viruses is the treatment by a combination of detergents, often including Triton X-100 (TX-100). Based on recent environmental concerns, the use of TX-100 in Europe will be ultimately banned, which forces the pharmaceutical industry, among others, to switch to an environmentally friendly alternative detergent with fully equivalent virus inactivation performance such as TX-100. In this study, a structure-activity relationship study was conducted that ultimately led to the synthesis of several new detergents. One of them, named "Nereid," displayed inactivation activity fully equivalent to TX-100. The synthesis of this replacement candidate has been optimized to allow for the production of several kg of detergent at lab scale, to enable the required feasibility and comparison virus inactivation studies needed to support a potential future transition. The 3-step, chromatography-free synthesis process described herein uses inexpensive starting materials, has a robust and simple work-up, and allows production in a standard organic laboratory to deliver batches of several hundred grams with >99% purity.

Reaction of Oxidized Polysialic Acid and a Diaminooxy Linker: Characterization and Process Optimization Using Nuclear Magnetic Resonance Spectroscopy.

Native polysialic acid (natPSA) is a high-molecular-weight glycan composed of repeat units of α-(2 → 8) linked N-acetylneuraminic acid (Neu5Ac). Mild periodate oxidation of PSA selectively targets the end sialic acid ring containing three adjacent alcohols generating a putative aldehyde, which can be used, after attachment of a linker molecule, for terminal attachment of PSA to protein. Previously, we showed that the oxidized PSA (oxoPSA) contained a hemiacetal at the oxidation site and can react with a linker containing an aminooxy group in a conjugation reaction to form a stable oxime linkage. Thus, reagents containing an aminooxy group may be prepared for conjugation of PSA to the carbohydrate moiety of therapeutic proteins, thereby increasing their half-life. These aminooxy-PSA reagents can selectively react with aldehyde groups generated by mild NaIO4 oxidation of glycans on the surface of the target protein. To comprehend the conjugation, unoxidized tetrasialic acid and Neu5Ac were reacted in model reactions with a diaminooxy linker to define the nuclear magnetic resonance (NMR) chemical shifts. Based on these data, we were able to show that, in the case of PSA, the reaction with the linker occurs not only at the expected oxidized end to form an aldoxime but also at the end distal to the oxidation to form a ketoxime. We determined that, in aged solutions, both oxoPSA and PSA aldoxime were hydrolyzed. PSA aldoxime was also shown to disproportionate to form a dimer (PSA-linker-PSA), which then could react further with the released linker at one of its PSA termini. Furthermore, NMR was used to monitor the effects of deliberate process changes so that conditions could be optimized for attachment of linker at the desired end of the PSA chain, which led to a well-defined product.