Sustainability Challenges and Opportunities in Oligonucleotide Manufacturing.

With a renewed and growing interest in therapeutic oligonucleotides across the pharmaceutical industry, pressure is increasing on drug developers to take more seriously the sustainability ramifications of this modality. With 12 oligonucleotide drugs reaching the market to date and hundreds more in clinical trials and preclinical development, the current state of the art in oligonucleotide production poses a waste and cost burden to manufacturers. Legacy technologies make use of large volumes of hazardous reagents and solvents, as well as energy-intensive processes in synthesis, purification, and isolation. In 2016, the American Chemical Society (ACS) Green Chemistry Institute Pharmaceutical Roundtable (GCIPR) identified the development of greener processes for oligonucleotide Active Pharmaceutical Ingredients (APIs) as a critical unmet need. As a result, the Roundtable formed a focus team with the remit of identifying green chemistry and engineering improvements that would make oligonucleotide production more sustainable. In this Perspective, we summarize the present challenges in oligonucleotide synthesis, purification, and isolation; highlight potential solutions; and encourage synergies between academia; contract research, development and manufacturing organizations; and the pharmaceutical industry. A critical part of our assessment includes Process Mass Intensity (PMI) data from multiple companies to provide preliminary baseline metrics for current oligonucleotide manufacturing processes.

Photocleavable peptide hydrogel arrays for MALDI-TOF analysis of kinase activity.

We have developed an acrylamide copolymerization strategy to immobilize acrylamide labeled peptides and proteins into a hydrogel surface and detect their modifications using MALDI-TOF mass spectrometry. Copolymerization into hydrogels is robust, compatible with "off-the-shelf" chemistry, and yields materials and surfaces that are stable to aqueous or organic solvents, drying, high or low temperature, high or low pH, oxidizing agents, sonication, mechanical contact, etc. The use of acrylamide hydrogels allows immobilization of substrates in a hydrated environment that can be used both as a biological reaction matrix and as a MALDI target. In our strategy, a substrate peptide was designed in a modular fashion to include both modification site and affinity domains. It was labeled with an acrylamide functionality using a generalized chemistry and covalently attached to the surface with a photocleavable linker, allowing for aggressive washing to remove any fouling, followed by selective release for MALDI-TOF analysis. Using this system we were able to analyze and compare v-Abl (truncated) and c-Abl (full-length) kinase activity on a peptide substrate with an affinity domain specific for the full-length kinase, observing excellent overall reproducibility in the extent of phosphorylation detected. This work serves as proof of principle for modular substrate design strategies for mass spectrometry-readable biosensors.

Protein-acrylamide copolymer hydrogels for array-based detection of tyrosine kinase activity from cell lysates.

We describe the development of an array-based assay for the molecular level detection of tyrosine kinase activity directly from cellular extracts. Glutathione S-transferase-Crkl (GST-Crkl) fusion proteins are covalently immobilized into polyacrylamide gel pads via copolymerization of acrylic monomer and acrylic-functionalized GST-Crkl protein constructs on a polyacrylamide surface. The resulting hydrogels resist nonspecific protein adsorption, permitting quantitative and reproducible determination of Abl tyrosine kinase activity and inhibition, even in the presence of a complex cell lysate mixture. Half-maximal inhibition (IC50) values for imatinib mesylate inhibition of GST-Crkl (SH3) phosphorylation by v-Abl in a purified system and Bcr-Abl within a K562 cell lysate were determined to be 1.5 and 20 microM, respectively. Additionally, the protein-acrylamide copolymer arrays detected CML cell levels as low as 15% in a background of Bcr-Abl- leukemic cells and provided the framework for the parallel evaluation of six tyrosine kinase inhibitors. Such a system may have direct application to the detection and treatment of cancers resulting from upregulated tyrosine kinase activity, such as chronic myeloid leukemia (CML). These findings also establish a basis for screening tyrosine kinase inhibitors and provide a framework on which protein-protein interactions in other complex systems can be studied.

Use of protein-acrylamide copolymer hydrogels for measuring protein concentration and activity.

We report the development and characterization of a polyacrylamide-based protein immobilization strategy for surface-bound protein assays, including concentration detection, binding affinity, and enzyme kinetics. Glutathione S-transferase (GST) fusion proteins have been labeled with an acrylic moiety and attached to acrylic-functionalized glass surfaces through copolymerization with acrylic monomer. The specific attachment of GST-green fluorescent protein (GFP) fusion protein was more than sevenfold greater than the nonspecific attachment of nonacrylic-labeled GST-GFP; 0.32 ng/mm(2) of surface-attached GST-GFP was detectable by direct measurement of GFP fluorescence and this lower detection limit was reduced to 0.080 ng/mm(2) using indirect antibody-based detection. The polyacrylamide-based surface attachment strategy was also used to measure the kinetics of substrate phosphorylation by the kinase c-Src. Michaelis-Menten kinetic constants for the reaction occurring in solution were K(m) = 2.7 +/- 1.0 microM and V(max) = 8.1 +/- 3.1 (arbitrary units). Kinetic values for the reaction utilizing surface-immobilized substrate were K(m) = 0.36 +/- 0.033 microM and V(max) = 9.7 +/- 0.63 and were found to be independent of the acrylamide concentration within the copolymer. Such a surface attachment strategy should be applicable to the proteomics field and addresses denaturation and dehydration problems associated with protein microarray development.